Chemically modified small molecules

ABSTRACT

Methods of modifying the rate of systemic absorption of a drug administered to a subject by a pulmonary route, the method comprising covalently conjugating a hydrophilic polymer to a drug, wherein the drug has a half-life of elimination from the lung of less than about 180 minutes, to form a drug-polymer conjugate, wherein the drug-polymer conjugate has a net hydrophilic character and a weight average molecular weight of from about 50 to about 20,000 Daltons, and wherein the half-life of elimination from the lung of the drug-polymer conjugate is at least about 1.5-fold greater than the half-life of elimination from the lung of the drug, wherein the half-life of elimination from the lung is measured by bronchoalveolar lavage followed by assaying residual lung material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 11/344,404, filed Jan. 30, 2006, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 11/015,196, filed Dec. 16, 2004, now U.S. Pat. No. 7,786,133, which claims priority to U.S. Provisional Patent Application No. 60/530,122, filed Dec. 16, 2003, all three applications of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention provides chemically modified small molecules and related methods that possess certain advantages over small molecules lacking the chemical modification. The chemically modified small molecules described herein relate to and/or have application(s) in the fields of drug discovery, pharmacotherapy, physiology, organic chemistry, polymer chemistry, and others.

BACKGROUND OF TILE INVENTION

The use of proteins as active agents has expanded in recent years due to several factors: improved techniques for identifying, isolating, purifying and/or recombinantly producing proteins; increased understanding of the roles of proteins in vivo due to the emergence of proteonomics; and improved formulations, delivery vehicles and approaches for chemically modifying proteins to enhance their pharmacokinetic or phamacodynamic properties. With respect to improved approaches for chemically modifying proteins, covalent attachment of a polymer such as poly(ethylene glycol) or PEG to a protein has been used to improve the circulating half-life, decrease immunogenicity, and/or reduce proteolytic degradation. This approach of covalently attaching PEG to a protein or other active agent is commonly referred to as PEGylation. Proteins for injection that are modified by covalent attachment of PEGs are typically modified by attachment of relatively high molecular weight PEG polymers that often range from about 5,000 to about 40,000 Daltons.

While modification of relatively large proteins for the purpose of improving their pharmaceutical utility is perhaps one of the most common applications of PEGylation, PEGylation has also been used, albeit to a limited degree, to improve the bioavailability and ease of formulation of small molecule drugs having poor aqueous solubilities. For instance, water-soluble polymers such as PEG have been covalently attached to artilinic acid to improve its aqueous solubility. See, for example, U.S. Pat. No. 6,461,603. Similarly, PEG has been covalently attached to triazine-based compounds such as trimelamol to improve their solubility in water and enhance their chemical stability. See, for example, International Patent Publication WO 02/043772. Covalent attachment of PEG to bisindolyl maleimides has been employed to improve poor bioavailability of such compounds due to low aqueous solubility. See, for example, International Patent Publication WO 03/037384. PEG chains attached to small molecule drugs for the purpose of increasing their aqueous solubility are typically of sizes ranging from about 500 Daltons to about 5000 Daltons, depending upon the molecular weight of the small molecule drug.

Active agents can be dosed by any of a number of administration routes including injection, oral, inhalation, nasal, and transdermal. One of the most preferred routes of administration, due to its ease, is oral administration. Oral administration, most common for small molecule drugs (i.e., non-protein-based drugs), is convenient and often results in greater patient compliance when compared to other routes of administration. Unfortunately, many small molecule drugs possess properties (e.g., low oral bioavailability) that render oral administration impractical. Often, the properties of small molecule drugs that are required for dissolution and selective diffusion through various biological membranes directly conflict with the properties required for optimal target affinity and administration. The primary biological membranes that restrict entrance of small molecule drugs into certain organs or tissues are membranes associated with certain physiological barriers, e.g., the blood-brain barrier, the blood-placental barrier, and the blood-testes barrier.

The blood-brain barrier protects the brain from most toxicants. Specialized cells called astrocytes possess many small branches, which form a barrier between the capillary endothelium and the neurons of the brain. Lipids in the astrocyte cell walls and very tight junctions between adjacent endothelial cells limit the passage of water-soluble molecules. Although the blood-brain barrier does allow for the passage of essential nutrients, the barrier is effective at eliminating the passage of some foreign substances and can decrease the rate at which other substances cross into brain tissue.

The placental barrier protects the developing and sensitive fetus from many toxicants that may be present in the maternal circulation. This barrier consists of several cell layers between the maternal and fetal circulatory vessels in the placenta. Lipids in the cell membranes limit the diffusion of water-soluble toxicants. Other substances such as nutrients, gases, and wastes of the developing fetus can, however, pass through the placental barrier. As in the case of the blood-brain barrier, the placental barrier is not totally impenetrable but effectively slows down the diffusion of many toxicants from the mother to the fetus in the art.

For many orally administered drugs, permeation across certain biological membranes such as the blood-brain barrier or the blood-placental barrier is highly undesirable and can result in serious side-effects such as neurotoxicity, insomnia, headache, confusion, nightmares or teratogenicity. These side effects, when severe, can be sufficient to halt the development of drugs exhibiting such undesirable brain or placental uptake.

U.S. Published Application No. 2003/0161791 A1, published Aug. 28, 2003, discloses water-soluble polymer conjugates of retinoic acid. The conjugates are prepared by covalent attachment of a water-soluble polymer such as polyethylene glycol to a retinoid such as retinoic acid. The conjugates are useful for inhalation therapy of conditions of the respiratory tract.

Thus, there is a need for new methods for effectively delivering drugs, and in particular small molecule drugs, to a patient while simultaneously reducing the adverse and often toxic side-effects of small molecule drugs. Specifically, there is a need for improved methods for delivering drugs that possess an optimal balance of good oral bioavailability, bioactivity, and pharmacokinetic profile. The present invention meets this and other needs.

SUMMARY OF THE INVENTION

The invention provides methods of modifying the rate of systemic absorption of a drug administered to a subject by a pulmonary route, the method comprising covalently conjugating a hydrophilic polymer to a drug, wherein the unconjugated drug has a half-life of elimination from the lung of less than about 180 minutes, to form a drug-polymer conjugate, wherein the drug-polymer conjugate has a net hydrophilic character and a weight average molecular weight of from about 50 to about 20,000 Daltons, and wherein the half-life of elimination from the lung of the drug-polymer conjugate is at least about 1.5-fold greater than the half-life of elimination from the lung of the unconjugated drug. In some embodiments, the half-life of elimination from the lung of the drug-polymer conjugate is at least about 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 500-fold greater than the half-life of elimination from the lung of the unconjugated drug.

In some embodiments, the hydrophilic polymer comprises a polymer chosen from polyethylene glycols and polyethylene oxides. In some embodiments, the weight average molecular weight of the polymer is from about 1000 to about 3500 Daltons. In some embodiments, the drug has a molecular weight of less than about 1500.

The hydrophilic polymer may comprise a polyethylene glycol, including for example, linear polyethylene glycols, branched polyethylene glycols, forked polyethylene glycols, and dumbbell polyethylene glycols. In some embodiments, the hydrophilic polymer comprises a polymer from a polydisperse population. In some embodiments, the hydrophilic polymer is a polymer chosen from monodisperse, bimodal, trimodal, or tetramodal polymer populations.

The invention also provides methods of controlling the lung residence time of a drug pulmonarily administered, comprising covalently attaching to the drug a hydrophilic polymer molecule having a weight average molecular weight of from about 50 to about 4000 Daltons, to form a drug-polymer conjugate. The hydrophilic polymer may be polyethylene glycol. The weight average molecular weight of the hydrophilic polymer is, in some embodiments, from about 1000 to about 3500 Daltons. In some embodiments, the drug has a molecular weight of less than about 1500. In some embodiments, the drug-polymer conjugate exhibits a net hydrophilic character.

The invention also provides methods of controlling the rate of systemic absorption of a drug pulmonarily administered comprising covalently attaching to the drug a hydrophilic polymer molecule having a weight average molecular weight of from about 50 to about 4000 Daltons, to form a drug-polymer conjugate.

The invention also provides pharmaceutical compounds for pulmonary administration comprising a drug covalently attached to a hydrophilic polymer, wherein the pharmaceutical compound has a net hydrophilic character, and wherein the weight average molecular weight of the hydrophilic polymer is from about 50 to about 3500 Daltons. In some embodiments, the invention provides compositions comprising the pharmaceutical compound according to the invention, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form, and in some embodiments, in dry form. The invention also provides aerosols comprising the composition according to the invention. The invention also provides inhaler devices including the compositions of the invention. In some embodiments, the compositions comprise particles having a mass median aerodynamic diameter (MMAD) of less than about 10 microns, or less than about 5 microns. In some embodiments, the composition comprises a dry powder. In some embodiments, the inhalers of the invention are characterized by an emitted dose of at least about 30 percent. The invention also provides spray-dried compositions.

In some embodiments of the invention, the hydrophilic polymer is covalently attached to the active ingredient molecule by a hydrolytically unstable linkage, which can be a linkage chosen from ester, thioester, and amide. In some embodiments, the hydrophilic polymer is covalently attached to the active ingredient by a hydrolytically stable linkage. In some embodiments, the hydrophilic polymer is polyethylene glycol, which may be chosen from linear polyethylene glycols, branched polyethylene glycols, forked polyethylene glycols, and dumbbell polyethylene glycols. The invention also provides unit dosage forms comprising the compositions according to the invention.

The invention also provides compounds according to the invention, wherein the weight average molecular weight of the hydrophilic polymer is from about 50 to about 3500 Daltons and wherein pulmonary administration of the compound results in a half-life of elimination from the lung that can be described by the equation, t_(1/2)−el=12.84*(1−e^(−kMW)), where k=0.000357, MW=molecular weight in Daltons, and t_(1/2)=elimination half-life in hours. The invention also provides methods of treating a systemic disease comprising pulmonarily administering the compounds according to the invention.

These and other objects, aspects, embodiments and features of the invention will become more fully apparent when read in conjunction with the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of necessary fee.

FIG. 1 is a plot of plasma concentration versus time for 13-cis retinoic acid (“13-cis-RA”) and exemplary small PEG conjugates thereof (PEG₃-13-cis retinamide, “PEG₃-13-cis RA”; PEG₅-13-cis retinamide, “PEG₅-13-cis RA; PEG₇-13-cis retinamide, “PEG₇-13-cis RA; and PEG₁₁-13-cis retinamide, “PEG₁₁-13-cis RA”) administered to Sprague Dawley rats as described in detail in Example 7.

FIG. 2 is a plot of plasma concentration versus time for 6-naloxol and exemplary small PEG conjugates thereof (3-mer, 5-mer, 7-mer) administered to Sprague Dawley rats as described in detail in Example 7.

FIG. 3 is a plot demonstrating the effect PEG chain length on the intestinal transport (as an indicator of oral bioavailability) of various PEG-13-cis-RA conjugates and 13-cis-RA in Sprague-Dawley rats.

FIG. 4 is a plot demonstrating the effect of covalent attachment of various sized PEG-mers on the blood-brain barrier transport of 13-cis-RA and various PEG-13-cis-RA conjugates.

FIG. 5 is a plot demonstrating the effect of covalent attachment of various sized PEG-mers on the intestinal transport (as an indicator of oral bioavailability) of naloxone and PEG_(n)-Nal.

FIG. 6 is a plot showing the effect of covalent attachment of various sized PEG-mers on the blood-brain barrier transport of naloxone and PEG-Nal.

FIG. 7 is a plot demonstrating the pharmacokinetics of naloxone and PEG_(n)-Nal in rats following oral gavage.

FIG. 8 and FIG. 9 are plots demonstrating the effect of covalent attachment of various sized PEG-mers on the level of naloxone metabolites and PEG_(n)-Nal metabolites.

FIG. 10 is mass spectrum of methoxy-PEG-350 obtained from a commercial source (Sigma-Aldrich). As can be seen from the analysis, although the reagent is sold as methoxy-PEG having a molecular weight of 350, the reagent is actually a mixture of nine distinct PEG oligomers, with the number of monomer subunits ranging from approximately 7 to approximately 15.

FIG. 11 diagrammatically shows the experimental strategy of Example 12.

FIG. 12 shows the elimination of PEG-FITC from BAL.

FIG. 13 shows the relationship between MW and Elimination half-life from BAL for a PEG-FITC conjugate.

FIG. 14 shows the uptake of PEG-FITC into BAL cells.

FIG. 15 shows the percentage dose of PEG-FITC that associates with the cellular fraction of BAL.

FIG. 16 shows the association of PEG-FITC with residual lung material.

FIG. 17 shows the concentration of PEG-FITC in serum.

FIGS. 18A and 18B show the total mass recovered in lung-derived fractions.

FIGS. 19A and 19B show the elimination of PEG-FITC from all combined lung compartments.

FIG. 20 shows in vitro permeability for Calu-3 cell studies.

FIG. 21 illustrates cell-based permeability plotted versus the in vivo absorption rate.

FIG. 22 shows the relationship between log P and PEG size for PEG-FITC conjugates.

FIG. 23 shows elimination from the lung for 2K PEG and 2K PEG-FITC.

FIG. 24 shows that increasing the dose of 2K PEG 10-fold does not significantly alter the elimination rate from the lung.

FIG. 25 shows the rate of disappearance from the lung of CIPRO and PEG-CIPRO conjugates.

FIG. 26 shows the rate in appearance of the plasma of CIPRO and PEG-CIPRO.

FIG. 27 shows the rate of appearance in the plasma of CIPRO.

FIG. 28 shows steroid-induced nuclear translocation assay of GR in CHO cells.

FIG. 29 shows the activation of Luciferase by TAA, PEG-3-TA, and PEG-7-TA.

FIG. 30 shows the time course activation of Luciferase by TAA.

FIG. 31 shows the difference in binding affinity between TA and the PEG-TA derivatives.

DETAILED DESCRIPTION OF THE INVENTION

It must be noted that, as used in this specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions described below.

“Water soluble” as in a “water-soluble oligomer” indicates an oligomer that is at least 35% (by weight) soluble, preferably greater than 95% soluble, and more preferably greater than 99% soluble, in water at room temperature at physiological pH (about 7.2-7.6). Typically, an unfiltered aqueous preparation of a “water-soluble” oligomer transmits at least 75%, more preferably at least 95%, of the amount of light transmitted by the same solution after filtering. On a weight basis, a “water soluble” oligomer is preferably at least 35% (by weight) soluble in water, more preferably at least 50% (by weight) soluble in water, still more preferably at least 70% (by weight) soluble in water, and still more preferably at least 85% (by weight) soluble in water. It is most preferred, however, that the water-soluble oligomer is at least 95% (by weight) soluble in water or completely soluble in water.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are used interchangeably herein and refer to one of the basic structural units of a polymer or oligomer. In the case of a homo-oligomer, this is defined as a structural repeating unit of the oligomer. In the case of a co-oligomer, a monomeric unit is more usefully defined as the residue of a monomer that was oligomerized to form the oligomer, since the structural repeating unit can include more than one type of monomeric unit. Preferred oligomers of the invention are homo-oligomers.

An “oligomer” is a molecule possessing from about 1 to about 30 monomers. The architecture of an oligomer can vary. Specific oligomers for use in the invention include those having a variety of geometries such as linear, branched, or forked, to be described in greater detail below. An oligomer is a type of polymer.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a “PEG oligomer” or an oligoethylene glycol is one in which all of the monomer subunits are ethylene oxide subunits. Typically, substantially all, or all, monomeric subunits are ethylene oxide subunits, though the oligomer may contain distinct end capping moieties or functional groups, e.g. for conjugation. Typically, PEG oligomers for use in the present invention will comprise one of the two following structures: “—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. As stated above, for the PEG oligomers of the invention, the variable (n) ranges from 1 to 30, and the terminal groups and architecture of the overall PEG can vary. When PEG further comprises a functional group, A, for linking to, e.g., a small molecule drug, the functional group when covalently attached to a PEG oligomer, does not result in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

An “end capping group” is generally a non-reactive carbon-containing group attached to a terminal oxygen of a PEG oligomer. For the purposes of the present invention, preferred are capping groups having relatively low molecular weights such as methyl or ethyl. The end-capping group can also comprise a detectable label. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric labels (e.g., dyes), metal ions, and radioactive moieties.

“Branched”, in reference to the geometry or overall structure of an oligomer, refers to an oligomer having two or more polymer “arms” extending from a branch point.

“Forked” in reference to the geometry or overall structure of an oligomer, refers to an oligomer having two or more functional groups (typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or more atoms at which an oligomer branches or forks from a linear structure into one or more additional arms.

The term “reactive” or “activated” refers to a functional group that reacts readily or at a practical rate under conventional conditions of organic synthesis. This is in contrast to those groups that either do not react or require strong catalysts or impractical reaction conditions in order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present on a molecule in a reaction mixture, indicates that the group remains largely intact under conditions effective to produce a desired reaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. The protecting group will vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule. Functional groups which may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like. Representative protecting groups for carboxylic acids include esters (such as a p-methoxybenzyl ester), amides and hydrazides; for amino groups, carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters; for thiol groups, thioethers and thioesters; for carbonyl groups, acetals and ketals; and the like. Such protecting groups are well-known to those skilled in the art and are described, for example, in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

A functional group in “protected form” refers to a functional group bearing a protecting group. As used herein, the term “functional group” or any synonym thereof is meant to encompass protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond is a relatively labile bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, thiolesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject to degradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, amines, and the like. Generally, a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater, more preferably 97% or greater, still more preferably 98% or greater, even more preferably 99% or greater, yet still more preferably 99.9% or greater, with 99.99% or greater being most preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantially all of the oligomers in the composition have a well-defined, single (i.e., the same) molecular weight and defined number of monomers, as determined by chromatography or mass spectrometry. Monodisperse oligomer compositions are in one sense pure, that is, substantially having a single and definable number (as a whole number) of monomers rather than a large distribution. A monodisperse oligomer composition of the invention possesses a MW/Mn value of 1.0005 or less, and more preferably, a MW/Mn value of 1.0000. By extension, a composition comprised of monodisperse conjugates means that substantially all oligomers of all conjugates in the composition have a single and definable number (as a whole number) of monomers rather than a large distribution and would possess a MW/Mn value of 1.0005 or less, and more preferably, a MW/Mn value of 1.0000 if the oligomer were not attached to the moiety derived from a small molecule drug. A composition comprised of monodisperse conjugates can, however, include one or more nonconjugate substances such as solvents, reagents, excipients, and so forth.

“Bimodal,” in reference to an oligomer composition, refers to an oligomer composition wherein substantially all oligomers in the composition have one of two definable and different numbers (as whole numbers) of monomers rather than a large distribution, and whose distribution of molecular weights, when plotted as a number fraction versus molecular weight, appears as two separate identifiable peaks. Preferably, for a bimodal oligomer composition as described herein, each peak is symmetric about its mean, although the size of the two peaks may differ. Ideally, the polydispersity index of each peak in the bimodal distribution, Mw/Mn, is 1.01 or less, more preferably 1.001 or less, and even more preferably 1.0005 or less, and most preferably a MW/Mn value of 1.0000. By extension, a composition comprised of bimodal conjugates means that substantially all oligomers of all conjugates in the composition have one of two definable and different numbers (as whole numbers) of monomers rather than a large distribution and would possess a MW/Mn value of 1.01 or less, more preferably 1.001 or less and even more preferably 1.0005 or less, and most preferably a MW/Mn value of 1.0000 if the oligomer were not attached to the moiety derived from a small molecule drug. A composition comprised of bimodal conjugates can, however, include one or more nonconjugated substances such as solvents, reagents, excipients, and so forth.

“Polydisperse” in reference to a polymer, refers to a composition having a polymer present in a distribution of molecular weights. The distribution generally will be a normal distribution, i.e., one that has a higher concentration of polymers with molecular weights near the mean, with a decrease in frequency as the difference from the mean molecular weight increases. The distribution may be a Gaussian distribution.

A “drug” is broadly used herein to refer to an organic, inorganic, or organometallic compound typically having a molecular weight of less than about 1500. Drugs of the invention encompass oligopeptides and other biomolecules having a molecular weight of less than about 1500. Peptide drugs of the invention have a molecular weight of less than about 1500 Daltons. It will be understood that the term “drug” refers to any drug in its active form, any prodrug, and any active ingredient. “Drug” as used herein includes any agent, compound, composition of matter or mixture which provides some pharmacologic, often beneficial, effect that can be demonstrated in vivo or in vitro. This includes foods, food supplements, nutrients, nutraceuticals, drugs, vaccines, antibodies, vitamins, and other beneficial agents. As used herein, these terms further include any physiologically or pharmacologically active substance that produces a localized or systemic effect in a patient. “Small molecule,” “small molecule drug,” and “drug” are used interchangeably herein.

The terms “moiety derived from a small molecule drug” and “small molecule drug moiety” are used interchangeably herein to refer to the portion or residue of the parent small molecule drug up to the covalent linkage resulting from covalent attachment of the drug (or an activated or chemically modified form thereof) to an oligomer of the invention.

A “biological membrane” is any membrane, typically made from specialized cells or tissues, that serves as a barrier to at least some xenobiotics or otherwise undesirable materials. As used herein a “biological membrane” includes those membranes that are associated with physiological protective barriers including, for example: the blood-brain barrier; the blood-cerebrospinal fluid barrier; the blood-placental barrier; the blood-milk barrier; the blood-testes barrier; and mucosal barriers including the vaginal mucosa, urethral mucosa, anal mucosa, buccal mucosa, sublingual mucosa, rectal mucosa, and so forth). Unless the context clearly dictates otherwise, the term “biological membrane” does not include those membranes associated with the middle gastro-intestinal tract (e.g., stomach and small intestines).

A “biological membrane crossing rate,” as used herein, provides a measure of a compound's ability to cross a biological barrier, such as the blood-brain barrier (“BBB”). A variety of methods can be used to assess transport of a molecule across any given biological membrane. Methods to assess the biological membrane crossing rate associated with any given biological barrier (e.g., the blood-cerebrospinal fluid barrier, the blood-placental barrier, the blood-milk barrier, the intestinal barrier, and so forth), are known, described herein and/or in the relevant literature, and/or can be determined by one of ordinary skill in the art.

A compound that “crosses the blood-brain barrier” in accordance with the invention is one that crosses the BBB at a rate greater than that of atenolol using the methods as described herein.

A “reduced rate of metabolism” in reference to the present invention, refers to a measurable reduction in the rate of metabolism of a water-soluble oligomer-small molecule drug conjugate as compared to rate of metabolism of the small molecule drug not attached to the water-soluble oligomer (i.e., the small molecule drug itself) or a reference standard material. In the special case of “reduced first pass rate of metabolism,” the same “reduced rate of metabolism” is required except that the small molecule drug (or reference standard material) and the corresponding conjugate are administered orally. Orally administered drugs are absorbed from the gastro-intestinal tract into the portal circulation and must pass through the liver prior to reaching the systemic circulation. Because the liver is the primary site of drug metabolism or biotransformation, a substantial amount of drug can be metabolized before it ever reaches the systemic circulation. The degree of first pass metabolism, and thus, any reduction thereof, can be measured by a number of different approaches. For instance, animal blood samples can be collected at timed intervals and the plasma or serum analyzed by liquid chromatography/mass spectrometry for metabolite levels. Other techniques for measuring a “reduced rate of metabolism” associated with the first pass metabolism and other metabolic processes are known, described herein and/or in the relevant literature, and/or can be determined by one of ordinary skill in the art. Preferably, a conjugate of the invention can provide a reduced rate of metabolism reduction satisfying at least one of the following values: at least about 5%, at least about 10%, at least about 15%; least about 20%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; and at least about 90%.

A compound (such as a small molecule drug or conjugate thereof) that is “orally bioavailable” is one that possesses a bioavailability when administered orally of greater than 1%, and preferably greater than 10%, where a compound's bioavailability is the fraction of administered drug that reaches the systemic circulation in unmetabolized form.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to 20 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl” includes cycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl

“Non-interfering substituents” are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substituted alkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, benzyl, etc.), preferably C₁-C₇.

“Electrophile” refers to an ion, atom, or an ionic or neutral collection of atoms having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or an ionic or neutral collection of atoms having a nucleophilic center, i.e., a center that is seeking an electrophilic center, and capable of reacting with an electrophile.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to an excipient that can be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

“Pharmacologically effective amount,” “physiologically effective amount,” and “therapeutically effective amount” are used interchangeably herein to mean the amount of a water-soluble oligomer-small molecule drug conjugate present in a composition that is needed to provide a desired level of active agent and/or conjugate in the bloodstream or in the target tissue. The precise amount will depend upon numerous factors, e.g., the particular active agent, the components and physical characteristics of the composition, intended patient population, patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein and available in the relevant literature.

A “difunctional” oligomer is an oligomer having two functional groups contained therein, typically at its termini. When the functional groups are the same, the oligomer is said to be homodifunctional. When the functional groups are different, the oligomer is said to be heterobifunctional.

A basic or acidic reactant described herein includes neutral, charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a conjugate as described herein, typically, but not necessarily, in the form of a water-soluble oligomer-small molecule drug conjugate, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

“Hydrophilic” in reference to a polymer or a drug-polymer conjugate means a compound or conjugate having a negative octanol:water partition coefficient (log P).

“Hydrophilic character” means being hydrophilic.

“Hydrolytically unstable” in reference to a linker means a linker that can be hydrolyzed under the conditions of a body.

“Residence time” means the amount of time a substance remains in a compartment—measured by half-life of elimination from that compartment.

“Rate of systemic absorption” means the rate at which a molecule crosses an epithelial layer to enter the systemic circulation.

The present invention is directed to (among other things) compositions of small molecule drugs that are chemically modified by covalent attachment of a water-soluble oligomer obtained from a monodisperse or bimodal composition of water-soluble oligomers. Because the water-soluble oligomer is often obtained from a monodisperse or bimodal composition of water-soluble oligomers, the resulting small molecule drug-oligomer compositions of the invention are typically exceedingly pure and well-defined from a structural standpoint.

An advantage of some of the conjugates described herein is their ability to exhibit a reduced biological membrane crossing rate as compared to the corresponding active agent not in conjugated form. While not wishing to be bound by theory, it is believed that molecular size is an important factor for determining whether and to what extent any given molecule can pass or cross any given biological membrane. For example, most if not all protective barriers, rely at least in part on highly packed cells that form a membrane having tight junctions through which only relatively small molecules can pass. Thus, for a given small molecule drug, the attachment of a water-soluble polymer to the small molecule drug provides a conjugate that is necessarily larger and with the expectation that the conjugate will either be prevented from crossing a biological membrane or will have a reduced biological membrane crossing rate as compared to the unconjugated small molecule drug.

As will be shown in further detail below and in the Experimental section, however, reducing the rate of biological membrane crossing by increasing molecular size by conjugating a water-soluble oligomer to a small molecule drug does not always provide a completely satisfactory conjugate. Often, the conjugate will be provided as a composition comprising monodisperse or bimodal conjugates. Again, while not wishing to be bound by theory, it is believed that even very small differences in the number of monomers between conjugates can provide relatively large differences in properties such as pharmacologic activity, metabolism, oral bioavailability, biological membrane crossing rate, solubility and others.

Furthermore, as is evidenced by the mass spectrum provided in FIG. 10, commercially available oligomer compositions such as PEG-350 are, in fact, relatively impure in that a range of oligomer sizes are present in the composition. Thus, the use of such relatively impure oligomer compositions (without further purification) in the synthesis of conjugates would result in a wide range of conjugate molecular weight sizes (as a result of the wide range of molecular weights in the composition used to form the conjugate). As a consequence, the resulting conjugate composition comprises many species of conjugates, wherein each conjugate would be expected to have different properties. From a regulatory and medicinal perspective, compositions comprising moieties having markedly different properties are ideally avoided.

As a result, in one or more embodiments, the present invention provides conjugates that are not only relatively large (as compared to the corresponding unconjugated small molecule drug) to reduce biological membrane crossing (again, as compared to the corresponding unconjugated small molecule drug), but are substantially pure as well to ensure consistent and desired activity and other properties of the composition. Thus, a composition is often provided comprising monodisperse or bimodal conjugates, each conjugate comprised of a moiety derived from a small molecule drug covalently attached by a stable linkage to a water-soluble oligomer, wherein said conjugate exhibits a reduced biological membrane crossing rate as compared to the biological membrane crossing rate of the small molecule drug not attached to the water-soluble oligomer.

As previously indicated, use of discrete oligomers from a well-defined composition of oligomers to form conjugates can advantageously alter certain properties associated with the corresponding small molecule drug. For instance, a conjugate of the invention, when administered by any of a number of suitable administration routes, such as parenteral, oral, transdermal, buccal, pulmonary, or nasal, exhibits reduced penetration across a biological membrane (such as the biological membranes associated with the blood-brain barrier and blood-placental barrier). It is preferred that the conjugates exhibit slowed, minimal or effectively no crossing of biological membranes (such as the biological membranes associated with the blood-brain barrier and blood-placental barrier), while still crossing the gastro-intestinal (GI) walls and into the systemic circulation if oral delivery is intended. The conjugates of the invention maintain a degree of bioactivity as well as bioavailability in their conjugated form.

In some embodiments in which pulmonary delivery is intended, the conjugate administered may have no crossing into systemic circulation or a reduced pulmonary tissue-blood barrier crossing rate so that local lung levels are maintained for local pharmacologic activity in the lung. Indeed, it has been surprisingly discovered that there is a direct relationship between the size of the polymer conjugated to the small molecule and its ability to slow the absorption from the lung. In some embodiments, an exponential relationship is observed from hydrophilic polymers, such as polyethylene glycol, having molecular weights of from about 50 Daltons to about 3500 Daltons. Thus, generally, as the molecular weight of the conjugated polymer increases from about 50 Daltons to about 3500 Daltons, so does the half-life of absorption through the lung. In some embodiments, a maximum ability to retard absorption is achieved by hydrophilic polymers having molecular weights of about 5000 Daltons, and additional increases in molecular weight may have little or no effect.

Still further, it has been surprisingly discovered that small molecules that are not normally readily absorbed through the lungs, can be caused to be absorbed, by their conjugation to hydrophilic polymers, according to the invention. While not wishing to be bound by any particular theory of operation, it appears that the conjugation to the hydrophilic polymer may cause the small molecule to be absorbed via a paracellular mechanism, as opposed to via a transcellular operation. This phenomenon may be a result of the imparted hydrophilicity, molecular size, or a combination of the two. Thus, small molecules that are normally taken up across the cell membranes of epithelial cells of the lungs are, according to this possible theory of operation, taken up through paracellular junctions. Again, for small molecules that are rapidly absorbed by the transcellular route, the conjugation according to the invention often results in a slower rate of absorption. And for small molecules that are very slowly absorbed, or even not absorbed at all, from the lung, a relative increase in the rate of absorption is often imparted by the present invention.

With respect to the blood-brain barrier (“BBB”), this barrier restricts the transport of drugs from the blood to the brain. This barrier consists of a continuous layer of unique endothelial cells joined by tight junctions. The cerebral capillaries, which comprise more than 95% of the total surface area of the BBB, represent the principal route for the entry of most solutes and drugs into the central nervous system.

Although it may be desirable for some compounds to achieve adequate concentrations in brain tissue to pharmacologically act therein, many other compounds that have no useful pharmacologic activity in brain tissue can ultimately reach the tissues of the central nervous system. By reducing the crossing rate of entry of these non-centrally acting compounds into the central nervous system, the risk of central nervous system side effects is reduced and the therapeutic effect may even be increased.

For compounds whose degree of blood-brain barrier crossing ability is not readily known, such ability can be determined using a suitable animal model such as an in situ rat brain perfusion (“RBP”) model as described herein. Briefly, the RBP technique involves cannulation of the carotid artery followed by perfusion with a compound solution under controlled conditions, followed by a wash out phase to remove compound remaining in the vascular space. (Such analyses can be conducted, for example, by contract research organizations such as Absorption Systems, Exton, Pa.). More specifically, in the RBP model, a cannula is placed in the left carotid artery and the side branches are tied off. A physiologic buffer containing the compound (5 micromolar) is perfused at a flow rate of 10 mL/min in a single pass perfusion experiment. After 30 seconds, the perfusion is stopped and the brain vascular contents are washed out with compound-free buffer for an additional 30 seconds. The brain tissue is then removed and analyzed for compound concentrations via liquid chromatograph with tandem mass spectrometry detection (LC/MS/MS). Alternatively, blood-brain barrier permeability can be estimated based upon a calculation of the compound's molecular polar surface area (“PSA”), which is defined as the sum of surface contributions of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule. The PSA has been shown to correlate with compound transport properties such as blood-brain barrier transport. Methods for determining a compound's PSA can be found, e.g., in, Ertl, P., et al., J. Med. Chem. 2000, 43, 3714-3717; and Kelder, J., et al., Pharm. Res. 1999, 16, 1514-1519.

A similar barrier to the blood-brain barrier is the blood-cerebrospinal fluid barrier. The blood-cerebrospinal fluid barrier creates a barrier or otherwise reduces the amount of toxic or undesirable substances reaching the cerebrospinal fluid, which is mostly located in the ventricular system and the subarachnoid space. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a patient can cross the blood-cerebrospinal fluid barrier, a known amount of the compound can be administered to mice by injection. A few days following administration of the compound, samples of mouse cerebrospinal fluid can be analyzed for the presence and amount of the compound.

The blood-placental barrier protects the developing fetus from most toxicants distributed in the maternal circulation. This barrier consists of several cellular layers between the maternal and fetal circulatory vessels in the placenta. As in the case of the blood-brain barrier, the placental barrier is not totally impenetrable but effectively slows down the diffusion of most toxicants. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a pregnant mammal can cross the blood-placental barrier, a known amount of the compound can be administered to pregnant mice by injection. A few days following administration of the compound, samples of mouse fetal tissue can be analyzed for the presence and amount of the compound.

The blood-milk barrier is similar to the blood-brain barrier in that a biological membrane separates and limits certain substances in the systemic circulation from crossing through. In the case of the blood-milk barrier, the biological membrane prevents certain substances from passing into the mammary glands. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a lactating mammal can cross the blood-milk barrier, a known amount of the compound can be administered to lactating mice by injection. A few days following administration of the compound, samples of milk from the mammary glands can be analyzed for the presence and amount of the compound.

The blood-testes barrier is comprised of sustentacular cells (Sertoli cells) cells which line the male reproductive tract and are joined by tight junctions. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a male mammal can cross the blood-testes barrier, a known amount of the compound can be administered to male mice by injection. A few days following administration of the compound, the mouse's testes can be removed and analyzed for the presence and amount of the compound.

Mucosal barriers represent another biological membrane that typically blocks or reduces undesirable substances from reaching systemic circulation. Administration of a compound to the particular mucosal area of interest and then analyzing a blood sample for the presence and amount of the compound can determine whether and to what extent the compound crosses that particular mucosal area.

With respect to any biological membrane, the water-soluble oligomer-small molecule drug conjugate exhibits a biological membrane crossing rate that is reduced as compared to the biological membrane crossing rate of the small molecule drug not attached to the water-soluble oligomer. Exemplary reductions in biological membrane crossing rates include reductions of: at least about 5%; at least about 10%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or at least about 90%, when compared to the biological membrane crossing rate of the small molecule drug not attached to the water-soluble oligomer. A preferred reduction in the biological membrane crossing rate for a conjugate is at least about 20%. In some instances, it is preferred that the small molecule drug itself is one that does cross one or more of the biological membranes described herein.

The conjugates exhibiting a reduced biological membrane crossing rate will typically comprise the structure O—X-D wherein: O corresponds to a water-soluble oligomer, X corresponds to a stable linkage, and D corresponds to the moiety derived from a small molecule drug.

The moiety derived from a small molecule drug is, in one sense, different than the parent small molecule drug in that it is linked, typically through a covalent bond, to an atom that is not associated with the parent small molecule drug. Except for the difference of being linked to another atom, however, the moiety derived from a small molecule drug is essentially the same as the small molecule drug and will have a similar pharmacologic mechanism of action. Thus, a discussion of the small molecule drug serves equally well to describe the moiety derived from a small molecule drug.

The active agents used in the conjugates are small molecule drugs, that is to say, pharmacologically active compounds typically having a molecular weight of less than about 1500 Daltons. Small molecule drugs, for the purpose of the invention, include oligopeptides, oligonucleotides, and other biomolecules having a molecular weight of less than about 1500 Daltons. Also encompassed in the term “small molecule drug” is any fragment of a peptide, protein, or antibody, including native sequences and variants falling within the molecular weight range stated above.

Exemplary molecular weights of small molecule drugs include molecular weights of: less than about 1400; less than about 1300; less than about 1200; less than about 1100; less than about 1000; less than about 950; less than about 900; less than about 850; less than about 800; less than about 750; less than about 700; less than about 650; less than about 600; less than about 550; less than about 500; less than about 450; less than about 400; less than about 350; and less than about 300.

The small molecule drug used in the invention, if chiral, may be obtained from a racemic mixture, or an optically active form, for example, a single optically active enantiomer, or any combination or ratio of enantiomers. In addition, the small molecule drug may possess one or more geometric isomers. With respect to geometric isomers, a composition can comprise only a single geometric isomer or a mixture of two or more geometric isomers. A small molecule drug for use in the present invention can be in its customary active form, or may possess some degree of modification. For example, a small molecule drug may have a targeting agent, tag, or transporter attached thereto, prior to or after covalent attachment of an oligomer. Alternatively, the small molecule drug may possess a lipophilic moiety attached thereto, such as a phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,” dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a small fatty acid. In some instances, however, it is preferred that the small molecule drug moiety does not include attachment to a lipophilic moiety.

A small molecule for use in coupling to an oligomer of the invention may be any of the following. Suitable agents may be selected from, for example, respiratory drugs, anticonvulsants, muscle relaxants, anti-inflammatories, appetite suppressants, antimigraine agents, muscle contractants, anti-infectives (antibiotics, antivirals, antifungals, vaccines) antiarthritics, antimalarials, antiemetics, bronchodilators, antithrombotic agents, antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants, anti-asthma agents, diuretics, lipid regulating agents, antiandrogenic agents, antiparasitics, anticoagulants, neoplastics, antineoplastics, hypoglycemics, nutritional agents and supplements, growth supplements, antienteritis agents, vaccines, antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number of structural classes, including but not limited to small molecules, oligopeptides, polypeptides or protein mimetics, fragments, or analogues, steroids, nucleotides, oligonucleotides, electrolytes, and the like. Preferably, an active agent for coupling to an oligomer of the invention possesses a free hydroxyl, carboxyl, thio, amino group, or the like (i.e., “handle”) suitable for covalent attachment to the oligomer. Alternatively, the drug is modified by introduction of a suitable “handle”, preferably by conversion of one of its existing functional groups to a functional group suitable for formation of a stable covalent linkage between the oligomer and the drug. Both approaches are illustrated in the Experimental section.

Specific examples of active agents suitable for covalent attachment to an oligomer of the invention include small molecule mimetics and active fragments (including variants) of the following: aspariginase, amdoxovir (DAPD), antide, becaplermin, calcitonins, cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO agonists (e.g., peptides from about 10-40 amino acids in length and comprising a particular core sequence as described in WO 96/40749), dornase alpha, erythropoiesis stimulating protein (NESP), coagulation factors such as Factor V, Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, von Willebrand factor; ceredase, cerezyme, alpha-glucosidase, collagen, cyclosporin, alpha defensins, beta defensins, exendin-4, granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen, filgrastim, growth hormones human growth hormone (hGH), growth hormone releasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bone morphogenic protein-2, bone morphogenic protein-6, OP-1; acidic fibroblast growth factor, basic fibroblast growth factor, levadopa, CD-40 ligand, heparin, human serum albumin, low molecular weight heparin (LMWH), interferons such as interferon alpha, interferon beta, interferon gamma, interferon omega, interferon tau, consensus interferon; interleukins and interleukin receptors such as interleukin-1 receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17 receptor; lactoferrin and lactoferrin fragments, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, insulin analogues (e.g., mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin, somatostatin analogs including octreotide, vasopressin, follicle stimulating hormone (FSH), influenza vaccine, insulin-like growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), plasminogen activators such as alteplase, urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growth factor (NGF), osteoprotegerin, platelet-derived growth factor, tissue growth factors, transforming growth factor-1, vascular endothelial growth factor, leukemia inhibiting factor, keratinocyte growth factor (KGF), glial growth factor (GGF), T Cell receptors, CD molecules/antigens, tumor necrosis factor (TNF), monocyte chemoattractant protein-1, endothelial growth factors, parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus antibody, cystic fibrosis transmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase), bactericidal/permeability increasing protein (BPI), and anti-CMV antibody. Exemplary monoclonal antibodies include etanercept (a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kD TNF receptor linked to the Fc portion of IgG1), abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomab tiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate, olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to an oligomer of the invention include but are not limited to amifostine, amiodarone, aminocaproic acid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide, anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide, bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cis retinoic acid, all trans retinoic acid; dacarbazine, dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, itraconazole, fluconazole, voriconazole, posiconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril, lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, metaraminol bitartrate, methotrexate, metoclopramide, mexiletine, mitomycin, mitotane, mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin, porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus, streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, dolasetron, granisetron; formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins, nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such as erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A; fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, and streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin, colistimethate; polymixins such as polymixin B, capreomycin, bacitracin, penems; penicillins including penicllinase-sensitive agents like penicillin G, penicillin V; penicllinase-resistant agents like methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram negative microorganism active agents like ampicillin, amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonal penicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams like aztreonam; and carbapenems such as imipenem, meropenem, pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasone diprepionate, triamcinolone acetamide, budesonide acetonide, fluticasone, ipratropium bromide, tiotropium, flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, and tyrphostines.

The above exemplary drugs are meant to encompass, where applicable, analogues, agonists, antagonists, inhibitors, isomers, polymorphs, and pharmaceutically acceptable salt forms thereof. Thus, for example, to the extent that an exemplary drug provided above is relatively large and would not be classified as a small molecule drug, the exemplary drug is still listed because an analogue of that large molecule having a similar activity but small size can be used.

Small molecule drugs particularly well suited for the invention are those that can measurably cross a biological membrane. Small molecule drugs exhibiting passage across the dermal barrier are also contemplated. In some instances, the small molecule drug is one, that when administered orally or even parenterally, undesirably crosses a biological barrier to a significant degree. For example, a small molecule drug that undesirably crosses the blood-brain barrier is one that exhibits a brain uptake rate greater than that of atenolol. In this regard, small molecule drugs that have a brain uptake rate (“BUR”), when measured as described herein, of greater than about 15 pmol/gm brain/sec are nonlimiting examples of small molecule drugs that undesirably cross the blood-brain barrier.

Thus, with respect to the blood-brain barrier, small molecule drugs intended for non-central nervous system indications that nonetheless cross the blood-brain barrier are preferred since conjugation of these drugs provides a molecule having less central nervous system side effects. For example, the structurally related nucleotides and nucleosides (e.g., 8-azaguanine, 6-mercaptupurine, azathioprene, thioinosinate, 6-methylthioinosinate, 6-thiouric acid, 6-thioguanine, vidarabine, cladribine, ancitabine, azacytidine, erythro-9-(2-hydroxy-3-nonyl)adenine, fludarabine, gemcitabine, and so forth) are preferred.

With respect to fludarabine, this small molecule drug exhibits about 70% oral bioavailability, and is used for treatment of chronic lymphocytic leukemia, as well as for treatment of hairy cell leukemia, non-Hodgkin's lymphoma, and mycosis fungoides. Fludarabine also exhibits central nervous system-related side effects, with severe neurologic effects including blindness, coma, and even death. Animal studies in rats and rabbits indicate that the drug may also be teratogenic. Thus, a fludarabine conjugate is expected to be effective in either blocking the penetration of drugs through the blood-brain barrier and/or blood-placenta barrier or at least slowing the crossing rate across these barriers such that adverse side effects of fludarabine are ameliorated.

Another class of small molecule drug that has common central nervous system-related side effects although is typically used for peripheral activities is the small molecule drug class of antihistamines. Structurally, antihistamines as a class are related as aminoalkyl ethers. Such small molecule drugs include diphenhydramine, bromodiphenhydramine, doxylamine, carbinoxamine, clemastine, dimenhydrinate, tripelennamine, pyrilamine, methapyrilene, thonzylamine, pheniramine, chlorpheniramine, dexchlorpheniramine, bromopheniramine, dexbromopheniramine, pyrrobutamine, triprolidine, promethazine, trimeprazine, methdilazine, cyclizine, chlorcyclizine, diphenylpyraline, phenindamine, dimethindene, meclizine, buclizine, antazoline, cyproheptadine, azatadine, terfenadine, fexofenadine, astemizole, cetirizine, azelastine, azatadine, loratadine, and desloratadine.

Still another class of small molecule drug in which a reduction in the blood-brain barrier crossing rate is desired are the opioid antagonists. Opioid antagonists include, naloxone, N-methylnaloxone, 6-amino-14-hydroxy-17-allylnordesomorphine, naltrendol, naltrexone, N-methylnaltrexone, nalbuphine, butorphanol, cyclazocine, pentazocine, nalmephene, naltrindole, nor-binaltorphimine, oxilorphan, 6-amino-6-desoxo-naloxone, pentazocine, levallorphanmethylnaltrexone, buprenorphine, cyclorphan, levalorphan, and nalorphine, as well as those described in U.S. Pat. Nos. 5,159,081, 5,250,542, 5,270,328, and 5,434,171 and in Knapp et al., “The pharmacology of Opioid Peptides” L. F. Tseng Ed., p. 15, Harwood Academic Publishers, 1995. Generally, however, any member of the oxymorphone chemical class (including the opioid antagonists above, as well as oxymorphone, codeine, oxycodone, morphine, ethylmorphine, diacetylmorphine, hydromorphone, dihydrocodeine, dihydromorphine, and methyldihydromorphine) is contemplated.

Another chemical class of small molecule drugs are the platinum coordination complex-based drugs. These include, for example, cisplatin, hydroplatin, carboplatin, and oxaliplatin.

Another class of small molecule drugs particularly well suited to be conjugated is the steroid class. Preferred steroids have a hydroxyl group in their molecular structure (or an acyl group that can be reduced to form a hydroxyl group). Nonlimiting examples of steroids include aldosterone, deoxycorticosterone, fludrocortisone, cortisone, hydrocortisone, prednisolone, prednisone, medrysone, meprednisone, alclometasone, beclomethasone, betamethasone, dexamethasone, diflorasone, flumethasone, methylprednisolone, paramethasone, amcinonide, desonide, budesonide, fluocinolone, flunisolide, flurandrenolide, triamcinolone, clobetasol, halcinonide, mometasone, clocortolone, and desoximetasone.

Fluoroquinolones and related small molecule drugs in this class can be used to form conjugates. Exemplary fluoroquinolones include those ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin and sitafloxacin.

Still another class of drug that is generally used for peripheral indications, some members of which are known to be teratogenic, is the retinoid class of small molecule drugs. The structurally related class of retinoids include, without limitation, retinol, retinal, 3-dehydroretinol, α-carotene, β-carotene, γ-carotene, δ-carotene, crytoxanthin, tretinoin, isotretinoin, etretinate, and eretin. Due to the potential for teratogenicity for this class of small molecule drug (or any class of drug that causes teratogenicity), it is desirable to reduce potential harm to the fetus by eliminating entirely or decreasing the rate of blood-placental barrier crossing of agents suspected of being teratogens.

Additional small molecule drugs for use as part of the conjugates described herein include phenothiazines, dibenzo-diazepines, galactogugues such as metoclopramide, and thiazides. Examples of phenothiazines include prochlorperazine, perphenazine, trifluoroperazine, and fluphenazine. Examples of dibenzo-diazepines include clozapine, olanzapine, and quetiapine. Other small molecule drugs include amlodipine, nifedipine, nimodipine, 5-hydroxytryptophan, retinoic acid, and isotretinoin. Another preferred drug is nevirapine, which readily crosses the placental barrier.

It should be noted that the small molecules referred to herein are provided as examples only, and moreover, that the listing of these small molecules does not imply that the invention has the same effect on all of these molecules. Obviously, each small molecule has its own physical-chemical characteristics that influence its contribution to the final characteristics of the complex. Furthermore, the listing of all of these small molecules together obviously does not mean that every one is useful for the treatment of the same condition or can be administered via the same route. Again, the specific qualities of the starting small molecule may contribute to the end use of the complex according to the invention. For at least these reasons, it is specifically noted that the listing of any class of small molecules, or even any small molecule within a class, implies the potential exclusion of that class, or specific small molecule, from particular embodiments of the invention. Thus, for example, while reference has been made herein to retinoids such as retinoic acid and isotretinoin, in some embodiments, that entire class of compounds, or specific molecules within that class, is excluded.

Additional small molecule drugs suitable for use in the invention can be found in, for example, in “The Merck Index, 13^(th) Edition, Merck & CO., Inc. (2001); “The AHFS Drug Handbook, 2^(nd) Edition”, American Society of Health System Pharmacists and Lippincott, Williams and Wilkins; “The Physicians Desk Reference”, Thomson Healthcare Inc., 2003; and “Remington: The Science and Practice of Pharmacy”, 19^(th) Edition, 1995.

By modifying the small drug molecule as provided above with covalent attachment of a water-soluble oligomer obtained from a monodisperse, bimodal, or polydisperse oligomer composition, significant changes in the small molecule drug's transport and pharmacological properties can result. The use of a water-soluble oligomer from a monodisperse, bimodal, or polydisperse oligomer composition allows for tailoring of drug properties, since the resultant conjugates form a well-defined composition rather than a distribution of a series of small molecule drug-oligomer conjugate species having a distribution of monomer subunits (and therefore molecular weights). As previously stated, the addition or deletion of as little as one monomer is observed to have a measurable effect on the properties of the resulting conjugate. Screening of a matrix of discrete oligomers of different sizes (from 1 to 30 monomer subunits) can be conducted in a reasonable amount of time, and allows for the tailoring of customizing of conjugates having optimized properties.

The oligomers, when attached to the small molecule drug, provide differences in properties compared to the parent small drug molecule. The use of small oligomers (in comparison to the 5K to 60K polymer chains that are typically attached to proteins) also increases the likelihood of the drug maintaining at least a degree, and preferably a significant degree, of its bioactivity. This feature is demonstrated in Table VI (Example 10), which provides bioactivity (EC₅₀) data for exemplary conjugates of the invention. The illustrative PEG oligomer-naloxone/naloxol conjugates possess bioactivities ranging from about 5% to about 35% of the unmodified parent drug, further demonstrating the beneficial features of the compounds of the invention.

The oligomer typically comprises two or more monomers serially attached to form a chain of monomers. The oligomer can be formed from a single monomer type (i.e., is homo-oligomeric) or two or three monomer types (i.e., is co-oligomeric). Preferably, each oligomer is a co-oligomer of two monomers or, more preferably, is a homo-oligomer. The monomer(s) employed result in an oligomer that is water soluble as defined herein.

Accordingly, each oligomer is typically composed of up to three different monomer types selected from the group consisting of: alkylene oxide, such as ethylene oxide or propylene oxide; olefinic alcohol, such as vinyl alcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkyl methacrylamide or hydroxyalkyl methacrylate, where alkyl is preferably methyl; α-hydroxy acid, such as lactic acid or glycolic acid; phosphazene, oxazoline, amino acids, carbohydrates such as monosaccharides, saccharide or mannitol; and N-acryloylmorpholine. Preferred monomer types include alkylene oxide, olefinic alcohol, hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, and α-hydroxy acid. Preferably, each oligomer is, independently, a co-oligomer of two monomer types selected from this group, or, more preferably, is a homo-oligomer of one monomer type selected from this group.

The two monomer types in a co-oligomer may be of the same monomer type, for example, two alkylene oxides, such as ethylene oxide and propylene oxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide. Usually, although not necessarily, the terminus (or termini) of the oligomer that is not covalently attached to a small molecule is capped to render it unreactive. Alternatively, the terminus may include a reactive group. When the terminus is a reactive group, the reactive group is either selected such that it is unreactive under the conditions of formation of the final oligomer or during covalent attachment of the oligomer to a small molecule drug, or it is protected as necessary. One common end-functional group is hydroxyl or —OH, particularly for oligoethylene oxides.

The water-soluble oligomer (“O” in the conjugate formula O—X-D) can have any of a number of different geometries. For example, “O” (in the formula O—X-D) can be linear, branched, or forked. Most typically, the water-soluble oligomer is linear or is branched, for example, having one branch point. Although much of the discussion herein is focused upon poly(ethylene oxide) as an illustrative oligomer, the discussion and structures presented herein can be readily extended to encompass any of the water-soluble oligomers described above.

The molecular weight of the water-soluble oligomer, excluding the linker portion, is generally relatively low. Exemplary values of the molecular weight of the water-soluble polymer include, in some embodiments: below about 5000; below about 4500; below about 4000; below about 3500; below about 3000; below about 2500; below about 2000; below about 1500; below about 1400; below about 1300; below about 1200; below about 1100; below about 1000; below about 900; below about 800; below about 700; below about 600; below about 500; below about 400; below about 300; below about 200; and below about 100 Daltons.

Exemplary ranges of molecular weights of the water-soluble oligomer (excluding the linker) include, in some embodiments: from about 100 to about 1400 Daltons; from about 100 to about 1200 Daltons; from about 100 to about 800 Daltons; from about 100 to about 500 Daltons; from about 100 to about 400 Daltons; from about 200 to about 500 Daltons; from about 200 to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75 to about 750 Daltons.

Preferably, the number of monomers in the water-soluble oligomer falls within one or more of the following ranges: between about 1 and about 30 (inclusive); between about 1 and about 25; between about 1 and about 20; between about 1 and about 15; between about 1 and about 12; between about 1 and about 10. In certain instances, the number of monomers in series in the oligomer (and the corresponding conjugate) is one of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, the oligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers in series. In yet further embodiments, the oligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series.

When the water-soluble oligomer has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers, these values correspond to a methoxy end-capped oligo(ethylene oxide) having a molecular weights of 75, 119, 163, 207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When the oligomer has 11, 12, 13, 14, or 15 monomers, these values correspond to methoxy end-capped oligo(ethylene oxide) having molecular weights corresponding to 515, 559, 603, 647, and 691 Daltons, respectively.

In those instances where a bimodal oligomer is employed, the oligomer will possess a bimodal distribution centering around any two of the above numbers of monomers. Ideally, the polydispersity index of each peak in the bimodal distribution, Mw/Mn, is 1.01 or less, and even more preferably, is 1.001 or less, and even more preferably is 1.0005 or less. Most preferably, each peak possesses a MW/Mn value of 1.0000. For instance, a bimodal oligomer may have any one of the following exemplary combinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth; 6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and 8-9, 8-10, and so forth.

In addition, the oligomer of the invention can be trimodal or even tetramodal, possessing a range of monomers units as previously described. Oligomer compositions possessing a well-defined mixture of oligomers (i.e., being bimodal, trimodal, tetramodal, etc.) can be prepared by mixing purified monodisperse oligomers to obtain a desired profile of oligomers (a mixture of two oligomers differing only in the number of monomers is bimodal; a mixture of three oligomers differing only in the number of monomers is trimodal; a mixture of four oligomers differing only in the number of monomers is tetramodal), or alternatively, can be obtained from column chromatography of a polydisperse oligomer by recovering the “center cut”, to obtain a mixture of oligomers in a desired and defined molecular weight range. As can be seen from FIG. 10, commercially available PEGs are typically polydisperse mixtures, even for low molecular weight materials. The methoxy-PEG sample shown was analyzed by mass spectrometry, and although labeled as methoxy-PEG-350, the reagent was found to contain 9 different PEG oligomer components, each differing in the number of monomer subunits. For the purposes of the present invention, that is to say, to prepare conjugates having the features described herein, polydisperse polymers are not particularly preferred, since small changes in the number of monomers have been discovered to have a profound effect on the properties of the resulting conjugates. Such effects would likely be dampened or even undetectable in a conjugate mixture prepared using a polydisperse oligomer. Moreover, commercial batches of polydisperse polymers (or oligomers) are often highly variable in their composition, and for this reason, are not particularly preferred for the present application, where batch-to-batch uniformity is a desirable feature for an oligomer as described herein.

As described above, the water-soluble oligomer is obtained from a composition that is preferably unimolecular or monodisperse. That is, the oligomers in the composition possess the same discrete molecular weight value rather than a distribution of molecular weights. Some monodisperse oligomers can be purchased from commercial sources such as those available from Sigma-Aldrich, or alternatively, can be prepared directly from commercially available starting materials such as Sigma-Aldrich. For example, oligoethylene glycols of the invention can be prepared as described, e.g., in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873 (1999), or in WO 02/098949 A1. Alternatively, such oligomers can be prepared as described herein in Example 9.

As described above, one aspect of the invention is an improved method of preparing a monodisperse oligomers such as an oligo(ethylene oxide). These oligomers can be used in any of a variety of applications, including but not limited to preparing a small molecule drug-water-soluble oligomer conjugate having the beneficial properties set forth above.

In order to provide the desired monodisperse oligomers, a new approach was used. It was discovered that halo-terminated oligomer reagents are more reactive and produce higher yields of monofunctional products in comparison to previously described reagents.

Thus, the present invention also includes a method for preparing monodisperse oligomer compositions. The method involves reacting a halo-terminated oligomer such as an oligo(ethylene oxide) having (m) monomers with a hydroxyl-terminated oligo(ethylene oxide) having (n) monomers. Generally, the halo group on the halo terminated oligoethylene glycol is a chloro, bromo or iodo group. Preferably, however, the halo group is bromo. The reaction is carried out under conditions effective to displace the halo group from the halo-terminated oligomer to thereby form an oligo(ethylene oxide) having (m)+(n) monomer subunits (OEG_(m+n)), where (m) and (n) each independently range from 1-10. That is to say, each of (m) and (n) is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, (m) and (n) each independently range from 1 to about 6. In selected embodiments, (m) is 1, 2, or 3 and (n) ranges from 1-6. In other instances, (m) is 1, 2, or 3, and (n) ranges from 2-6. Typically, the reaction is carried out in the presence of a strong base effective to convert the hydroxyl group of the hydroxyl-terminated oligoethylene oxide into the corresponding alkoxide species. Suitable bases include sodium, potassium, sodium hydride, potassium hydride, sodium methoxide, potassium methoxide, sodium tert-butoxide, and potassium tert-butoxide. In a preferred embodiment, the halo-terminated oligoethylene glycol possesses an end-capping group such as methoxy or ethoxy.

Representative hydroxy-terminated oligo(ethylene glycol)s correspond to the structure HO—(CH₂CH₂O)_(n)—H, where (n) is as described above. The method then preferably includes the step of converting the terminal hydroxyl group of OEG_(m+n) into a halo group, —X, to form OEG_(m+n)-X. The above steps are then repeated until a unimolecular oligomer having the desired number of subunits is obtained.

An illustrative reaction scheme is as follows.

As shown, the method involves the coupling of two unimolecular oligomer species by employing a substitution reaction where a halide on one oligomer, preferably an oligomeric ethylene oxide, and even more preferably, a halo-derivatized oligoethylene oxide methyl ether, is reacted with an oligoethylene glycol-alkoxide to generate the corresponding oligomer (see reaction 1 above).

The alkoxide is typically generated from the corresponding oligoethylene oxide by converting the terminal hydroxyl to the corresponding alkoxide in the presence of a strong base. The reaction is generally carried out in an organic solvent such as tetrahydrofuran (“THF”) at temperatures ranging from about 0° C. to about 80° C. Reaction times typically range from about 10 minutes to about 48 hours. The resultant product, in the exemplary reaction above, an end-capped oligoethylene oxide, contains a sum of the number of monomers of the halo-derivatized oligomer and the number of monomers in the oligoethylene glycol alkoxide [(m)+(n)]. Yields typically range from about 25% to about 75% for the purified coupled product, with yields most typically ranging from about 30 to about 60%.

In the above example, the hydroxyl terminus in the product from reaction 1 is then activated, if necessary, for coupling to a small molecule. Alternatively, if desired, the hydroxyl terminus in the exemplary product shown above [in the above example having (m)+(n) subunits), is then converted to a halide, preferably a bromide. Conversion of an alcohol to an alkyl halide can by effected directly, or through an intermediate such as a sulfonate or haloformate. Conditions and reagents suitable for effecting this transformation are found, for example, in Larock, R., “Comprehensive Organic Transformations”, VCH, 1994, pages 353 to 363.

One preferred method is that set forth in Example 11. The stepwise addition of the oligoethylene oxide halide to an oligoethylene oxide is then repeated as described above, to form an oligoethylene oxide having (m)+2(n) monomers, and so-forth. In this manner, discrete oligoethylene oxide subunits are then added in a controlled, stepwise fashion to the existing monomeric (unimolecular) oligomeric ethylene oxide product, to ensure preparation of a well-defined oligomer having an exact number of subunits.

Commonly available are unimolecular oligoethylene glycols having from about 1-3 monomer subunits (Sigma-Aldrich). Use of a halo-substituted oligomeric ethylene glycol reactant represents an improvement over existing methods, e.g., employing the mesylate, since the approach provided herein results in improved yields, shorter reaction times and milder reaction conditions due to the higher reactivity of the halide, and in particular, the bromo-substituted oligoethylene glycol reagent. Oligomers thus prepared are typically purified prior to further use, for example, by one or more of the following methods: chromatography such as HPLC, ion exchange chromatography, column chromatography, precipitation, or recrystallization. Purity is then confirmed by any of a number of analytical techniques, such as NMR, GPC, and FTIR. Products thus formed are then suitable for further use.

The linker or linkage of the invention may be a single atom, such as an oxygen or a sulfur, two atoms, or a number of atoms. A linker is typically but is not necessarily linear in nature. The linkage, “X” (in the O—X-D formula), is hydrolytically stable, and is preferably also enzymatically stable. Preferably, the linkage “X” is one having a chain length of less than about 12 atoms, and preferably less than about 10 atoms, and even more preferably less than about 8 atoms or even more preferably less than about 5 atoms, whereby length is meant the number of atoms in a single chain, not counting substituents. For instance, a urea linkage such as this, R_(oligomer)—NH—(C═O)—NH—R′_(drug), is considered to have a chain length of 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkage does not comprise further spacer groups. Small linkages are preferred and lend themselves to the nature of the present invention, since small linkages such as these are less likely to dominate or overshadow the effect of an addition of one or a small number of monomer subunits on the difference in transport properties of the conjugates of the invention.

In some instances, the linker “X” is hydrolytically stable and comprises an ether, amide, urethane, amine, thioether, urea, or a carbon-carbon bond. Functional groups such as those discussed below, and illustrated in the working examples, are typically used for forming the linkages. The linkage may less preferably also comprise (or be adjacent to or flanked by) spacer groups, as described further below. Spacers are most useful in instances where the bioactivity of the conjugate is significantly reduced due to the positioning of the oligomer on the parent drug.

More specifically, in selected embodiments, a linker of the invention, L, may be any of the following: —O—, —NH—, —S—, —C(O)—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group, —N(R⁶)—, R⁶ is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a series of atoms is not considered as a linkage when the series of atoms is immediately adjacent to an oligomer segment, and the series of atoms is but another monomer such that the proposed linkage would represent a mere extension of the oligomer chain.

The linkage “X” between the oligomer and the small molecule is typically formed by reaction of a functional group on a terminus of the oligomer with a corresponding functional group within the small molecule drug. Illustrative reactions are described briefly below. For example, an amino group on an oligomer, “O,” may be reacted with a carboxylic acid or an activated carboxylic acid derivative on the small molecule, or vice versa, to produce an amide linkage. Alternatively, reaction of an amine on an oligomer with an activated carbonate (e.g. succinimidyl or benzotriazyl carbonate) on the drug, or vice versa, forms a carbamate linkage. Reaction of an amine on an oligomer with an isocyanate (R—N═C═O) on a drug, or vice versa, forms a urea linkage (R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group on an oligomer with an alkyl halide, or halide group within a drug, or vice versa, forms an ether linkage. In yet another coupling approach, a small molecule having an aldehyde function is coupled to an oligomer amino group by reductive amination, resulting in formation of a secondary amine linkage between the oligomer and the small molecule.

A particularly preferred oligomer is an oligomer bearing an aldehyde functional group. In this regard, the oligomer will have the following structure: CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred (n) values include 3, 5, and 7, and preferred (p) values include 2, 3, and 4. In addition, the carbon atom alpha to the —C(O)H moiety can optionally be substituted with alkyl. The oligomer reagent is preferably provided as a monodisperse composition.

Typically, the terminus of the oligomer not bearing a functional group is capped to render it unreactive. When the oligomer does includes a further functional group at a terminus other than that intended for formation of a conjugate, that group is either selected such that it is unreactive under the conditions of formation of the linkage “X,” or it is protected during the formation of the linkage “X.”

As stated above, the oligomer includes a functional group for forming a small molecule conjugate having the properties described herein. The functional group typically comprises an electrophilic or nucleophilic group for covalent attachment to a small molecule, depending upon the reactive group contained within or introduced into the small molecule. Examples of nucleophilic groups that may be present in either the oligomer or the small molecule include hydroxyl, amine, hydrazine (—NHNH₂), hydrazide (—C(O)NHNH₂), and thiol. Preferred nucleophiles include amine, hydrazine, hydrazide, and thiol, particularly amine. Most small molecule drugs for covalent attachment to an oligomer will possess a free hydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present in either the oligomer or the small molecule include carboxylic acid, carboxylic ester, particularly imide esters, orthoester, carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy, sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. More specific examples of these groups include succinimidyl ester or carbonate, imidazoyl ester or carbonate, benzotriazole ester or carbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyl disulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, and tresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such as thione, thione hydrate, thioketal, etc., as well as hydrates or protected derivatives of any of the above moieties (e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal, thioketal, thioacetal). Another useful conjugation reagent is 2-thiazolidine thione.

As noted above, an “activated derivative” of a carboxylic acid refers to a carboxylic acid derivative which reacts readily with nucleophiles, generally much more readily than the underivatized carboxylic acid. Activated carboxylic acids include, for example, acid halides (such as acid chlorides), anhydrides, carbonates, and esters. Such esters include imide esters, of the general form —(CO)O—N[(CO)—]₂; for example, N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Also preferred are imidazolyl esters and benzotriazole esters. Particularly preferred are activated propionic acid or butanoic acid esters, as described in co-owned U.S. Pat. No. 5,672,662. These include groups of the form —(CH₂)₂₋₃(C═O)O-Q, where Q is preferably selected from N-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole, 7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate, maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate, p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyl disulfide.

These electrophilic groups are subject to reaction with nucleophiles, e.g. hydroxy, thio, or amino groups, to produce various bond types. Preferred for the present invention are reactions which favor formation of a hydrolytically stable linkage. For example, carboxylic acids and activated derivatives thereof, which include orthoesters, succinimidyl esters, imidazolyl esters, and benzotriazole esters, react with the above types of nucleophiles to form esters, thioesters, and amides, respectively, of which amides are the most hydrolytically stable. As mentioned above, most preferred are conjugates having a hydrolytically stable linkage between the oligomer and the drug. Carbonates, including succinimidyl, imidazolyl, and benzotriazole carbonates, react with amino groups to form carbamates. Isocyanates (R—N═C═O) react with hydroxyl or amino groups to form, respectively, carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes, ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, and ketal) are preferably reacted with amines, followed by reduction of the resulting imine, if desired, to provide an amine linkage (reductive amination).

Several of the electrophilic functional groups include electrophilic double bonds to which nucleophilic groups, such as thiols, can be added, to form, for example, thioether bonds. These groups include maleimides, vinyl sulfones, vinyl pyridine, acrylates, methacrylates, and acrylamides. Other groups comprise leaving groups which can be displaced by a nucleophile; these include chloroethyl sulfone, pyridyl disulfides (which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate, thiosulfonate, and tresylate. Epoxides react by ring opening by a nucleophile, to form, for example, an ether or amine bond. Reactions involving complementary reactive groups such as those noted above on the oligomer and the small molecule are utilized to prepare the conjugates of the invention.

For instance, the preparation of an exemplary oligomeric conjugate of retinoic acid is described in detail in Example 1. Briefly, the small molecule, retinoic acid, which contains a reactive carboxyl group, is coupled to an amino-activated oligomeric ethylene glycol, to provide a conjugate having an amide group covalently linking the small molecule to the oligomer. The covalent attachment of each a PEG 3-mer (meaning an oligomeric ethylene glycol having 3 ethylene glycol monomer subunits), a PEG 7-mer, and a PEG 11-mer to retinoic acid is described.

Further, the preparation of an oligomer-conjugate of naloxone is described in Example 4. In this representative synthesis, following protection of an aromatic hydroxyl group, a keto group in naloxone is reduced to the corresponding hydroxyl, which is then coupled to an oligomeric ethylene glycol halide to result in an ether (—O—) linked small molecule conjugate. Interestingly, in this example, reduction of the hydroxyl group in naloxone resulted in formation of two stereoisomers differing in the orientation of the hydroxyl group. The corresponding oligomeric conjugates were prepared and separated, and shown to have somewhat different characteristics, to be discussed in greater detail below. This represents another feature of the invention, that is, the preparation/isolation of single isomers of oligomer-small molecule conjugates, and uses thereof.

The conjugates of the invention exhibit a reduced biological barrier crossing rate as previously described. Moreover, the conjugates typically maintain at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more of the bioactivity of the unmodified parent small molecule drug. For a given small molecule drug having more than one reactive site suitable for modification, it may be necessary to carry out molecular modeling, or in vivo or in vitro biological activity assays to assess the biological activity of the resulting conjugate and determine the site most suitable for covalent attachment of an oligomer. See for example the illustrative bioactivity data in Table VI for various oligomer conjugates of naloxone and derivatized naloxone, 6-NH₂-naloxone and 6-OH-naloxol. In this investigation, variables included the site of chemical modification on the parent drug, type of covalent linkage, stereochemistry, and size of oligomer covalently attached to the drug moiety. As can be seen from the data, the bioactivities of the conjugates ranged from about 5% to about 35% of the bioactivity of the parent drug.

It has been discovered that stable covalent attachment of small, water-soluble oligomers to orally bioavailable small molecule drugs is effective to significantly alter the properties of these molecules, thereby making them more clinically effective. More specifically, covalent attachment of monodisperse oligomers such as oligoethylene oxide is often effective to reduce, or in some cases, eliminate, a drug's transport across the blood brain barrier, which then translates into a significant reduction in central nervous system-related side effects. The selection of an optimally sized oligomer can be conducted as follows.

First, an oligomer obtained from a monodisperse or bimodal water soluble oligomer is conjugated to a small molecule drug. Preferably, the drug is orally bioavailable, and on its own, exhibits a biological membrane crossing rate. Next, the ability of the conjugate to cross the biological membrane is determined using an appropriate model and compared to that of the unmodified parent drug. If the results are favorable, that is to say, if, for example, the rate of crossing is significantly reduced, then the bioactivity of conjugate is further evaluated. A beneficial conjugate in accordance with the invention is bioactive, since the linkage is hydrolytically stable and does not result in release of unmodified drug upon administration. Thus, the drug in conjugated form should be bioactive, and preferably, maintains a significant degree of bioactivity relative to the parent drug, i.e., greater than about 30% of the bioactivity of the parent drug, or even more preferably, greater than about 50% of the bioactivity of the parent drug. Then, the above steps are repeated using oligomers of the same monomer type but having a different number of subunits.

Because the gastro-intestinal tract (“GIT”) limits the transport of food and drugs from the digestive lumen in to blood and the lymph, the GIT represents another barrier for which the conjugate may be tested. The GIT barrier, however, represents a barrier that must not block the conjugates when the conjugate is intended for oral administration for systemic delivery. The GIT barrier consists of continuous layers of intestinal cells joined by tight junctions in the intestinal epithelia.

For each conjugate whose ability to cross a biological membrane is reduced in comparison to the non-conjugated small molecule drug, its oral bioavailability is then assessed. Based upon these results, that is to say, based upon the sequential addition of increasing numbers of discrete monomers to a given small molecule at a given position or location within the small molecule, it is possible to determine the size of the oligomer most effective in providing a conjugate having an optimal balance between reduction in biological membrane crossing, oral bioavailability, and bioactivity. The small size of the oligomers makes such screenings feasible, and allows one to effectively tailor the properties of the resulting conjugate. By making small, incremental changes in oligomer size, and utilizing an experimental design approach, one can effectively identify a conjugate having a favorable balance of reduction in biological membrane crossing rate, bioactivity, and oral bioavailability. In some instances, attachment of an oligomer as described herein is effective to actually increase oral bioavailability of the drug.

In view of the present disclosure, one of ordinary skill in the art, using routine experimentation, can determine a best-suited molecular size and linkage for improving oral bioavailability by first preparing a series of oligomers with different weights and functional groups and then obtaining the necessary clearance profiles by administering the conjugates to a patient and taking periodic blood and/or urine sampling. Once a series of clearance profiles have been obtained for each tested conjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) can also be used to study oral drug transport. In addition, non-in vivo methods include rodent everted gut excised tissue and Caco-2 cell monolayer tissue-culture models. These models are useful in predicting oral drug bioavailability.

The present invention also includes pharmaceutical preparations comprising a conjugate as provided herein in combination with a pharmaceutical excipient. Generally, the conjugate itself will be in a solid form (e.g., a precipitate), which can be combined with a suitable pharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

The preparation may also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA, zinc and other such suitable cations.

Acids or bases may be present as an excipient in the preparation. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.

Generally, however, the excipient will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5%-98% by weight, more preferably from about 15-95% by weight of the excipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipients are described in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed., Williams & Williams, (1995), the “Physician's Desk Reference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition, American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and the invention is not limited in this regard. Exemplary preparations are most preferably in a form suitable for oral administration such as a tablet, caplet, capsule, gel cap, troche, dispersion, suspension, solution, elixir, syrup, lozenge, transdermal patch, spray, suppository, and powder.

Oral dosage forms are preferred for those conjugates that are orally active, and include tablets, caplets, capsules, gel caps, suspensions, solutions, elixirs, and syrups, and can also comprise a plurality of granules, beads, powders or pellets that are optionally encapsulated. Such dosage forms are prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts.

Tablets and caplets, for example, can be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred when preparing tablets or caplets containing the conjugates described herein. In addition to the conjugate, the tablets and caplets will generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, coloring agents, and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose and lactose), polyethylene glycol, waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, microcrystalline cellulose, ethyl cellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricants are used to facilitate tablet manufacture, promoting powder flow and preventing particle capping (i.e., particle breakage) when pressure is relieved. Useful lubricants are magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums, or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, and microcrystalline cellulose, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, and sorbitol. Stabilizers, as well known in the art, are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions.

Capsules are also preferred oral dosage forms, in which case the conjugate-containing composition can be encapsulated in the form of a liquid or gel (e.g., in the case of a gel cap) or solid (including particulates such as granules, beads, powders or pellets). Suitable capsules include hard and soft capsules, and are generally made of gelatin, starch, or a cellulosic material. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (typically as a lyophilizate or precipitate, which can be in the form of a powder or cake), as well as formulations prepared for injection, which are typically liquid and requires the step of reconstituting the dry form of parenteral formulation. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration can take the form of nonaqueous solutions, suspensions, or emulsions, each typically being sterile. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.

The parenteral formulations described herein can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. The formulations are rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat.

The conjugate can also be administered through the skin using conventional transdermal patch or other transdermal delivery system, wherein the conjugate is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the conjugate is contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated structure can contain a single reservoir, or it can contain multiple reservoirs.

The pulmonary formulations may take various forms. Examples of pharmaceutically acceptable excipients for pulmonary delivery include, but are not limited to, lipids, metal ions, surfactants, amino acids, peptides, carbohydrates, buffers, salts, polymers, and the like, and combinations thereof. Vehicles for pulmonary delivery and processes of making such vehicles are disclosed, e.g., in U.S. Pat. Nos. 6,518,239 and 6,565,885, which are incorporated herein by reference.

The invention also provides a method for administering a conjugate as provided herein to a patient suffering from a condition that is responsive to treatment with the conjugate. The method comprises administering, generally orally, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical preparation). Other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal, and parenteral. As used herein, the term “parenteral” includes subcutaneous, intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injection.

In instances where parenteral administration is utilized, it may be necessary or desirable to employ somewhat larger oligomers than those described previously, with molecular weights ranging from about 500 to 30K Daltons (e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that can be remedied or prevented by administration of the particular conjugate. Those of ordinary skill in the art appreciate which conditions a specific conjugate can effectively treat. The actual dose to be administered will vary depend upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature. Generally, a therapeutically effective amount will range from about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.

One advantage of administering the conjugates of the present invention is that a reduction in first pass metabolism may be achieved relative to the parent drug. See for example the supporting results in Example 8. Such a result is advantageous for many orally administered drugs that are substantially metabolized by passage through the gut. In this way, clearance of the conjugate can be modulated by selecting the oligomer molecular size, linkage, and position of covalent attachment providing the desired clearance properties. One of ordinary skill in the art can determine the ideal molecular size of the oligomer based upon the teachings herein. All articles, books, patents, patent publications and other publications referenced herein are incorporated by reference in their entireties. Preferred reductions in first pass metabolism for a conjugate as compared to the corresponding nonconjugated small drug molecule include: at least about 10%, at least about 20%, at least about 30; at least about 40; at least about 50%; at least about 60%, at least about 70%, at least about 80% and at least about 90%.

Thus, the invention provides a method for reducing the metabolism of an active agent. The method comprises: providing monodisperse or bimodal conjugates, each conjugate comprised of a moiety derived from a small molecule drug covalently attached by a stable linkage to a water-soluble oligomer, wherein said conjugate exhibits a reduced rate of metabolism as compared to the rate of metabolism of the small molecule drug not attached to the water-soluble oligomer; and administering the conjugate to a patient. Typically, administration is carried out via one type of administration selected from the group consisting of oral administration, transdermal administration, buccal administration, transmucosal administration, vaginal administration, rectal administration, parenteral administration, and pulmonary administration.

Although useful in reducing many types of metabolism (including both Phase I and Phase II metabolism), the conjugates are particularly useful when the small molecule drug is metabolized by a hepatic enzyme (e.g., one or more of the cytochrome P450 isoforms) and/or by one or more intestinal enzymes.

In accordance with some embodiments of the present invention, methods of modifying the route of absorption of a compound administered to a subject by a pulmonary route are provided. The methods comprise covalently attaching a hydrophilic polymer to a compound to form a compound-polymer conjugate. In one example, the compound-polymer conjugate has a net hydrophilic character.

The conjugates exhibiting modified routes of absorption of a compound can comprise the structure: P-L-D, wherein P comprises a hydrophilic polymer, L comprises a linker, and D comprises a drug. In accordance with embodiments of the invention, the polymer P can be chosen from any of the water soluble oligomers O that exhibit hydrophilic character described herein. In one embodiment, the polymer P can be chosen from a monodisperse or bimodal population of oligomers as described herein with respect with to the water soluble oligomers O. In other embodiments, the polymers can be chosen from a polydisperse population of polymers comprising hydrophilic oligomers O of varying molecular weights. In one embodiment, the polymer P can be a polydisperse population of polyethylene glycols (PEGs). In another embodiment, the PEG can be a monodisperse or bimodal PEG population. The PEG can have any suitable geometry. For example, the PEG can be a linear PEG, branched PEG, forked PEG, and a dumbbell PEG, and combinations thereof.

In instances in which pulmonary administration is utilized, the molecular weight may be less than about 5000 Daltons, or less than about 4000 Daltons, or less than about 3000 Daltons, or less than about 2000 Daltons, or less than about 1000 Daltons. In some embodiments, the ranges of molecular weights for pulmonary administration will range from about 50 Daltons to about 3500 Daltons. However, the molecular weights of the polymers are not so limited. The polymer P can have a weight average molecular weight of from about x to about y, wherein x and y include, but are not limited to: 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350.8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, 13000, 13050, 13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, 15000, 15000, 15050, 15100, 15150, 15200, 15250, 15300, 15350, 15400, 15450, 15500, 15550, 15600, 15650, 15700, 15750, 15800, 15850, 15900, 15950, 16000, 16050, 16100, 16150, 16200, 16250, 16300, 16350, 16400, 16450, 16500, 16550, 16600, 16650, 16700, 16750, 16800, 16850, 16900, 16950, 17000, 17050, 17100, 17150, 17200, 17250, 17300, 17350, 17400, 17450, 17500, 17550, 17600, 17650, 17700, 17750, 17800, 17850, 17900, 17950, 18000, 18050, 18100, 18150, 18200, 18250, 18300, 18350, 18400, 18450, 18500, 18550, 18600, 18650, 18700, 18750, 18800, 18850, 18900, 18950, 19000, 19050, 19100, 19150, 19200, 19250, 19300, 19350, 19400, 19450, 19500, 19550, 19600, 19650, 19700, 19750, 19800, 19850, 19900, 19950, and 20000 Daltons. Thus, in one example, the weight average molecular weight of the polymer P is between about 50 and about 20000 Daltons. In another example, the weight average molecular weight of the polymer P is between about 50 and about 5000 Daltons, or between about 1000 and about 3500 Daltons. In yet another example, the weight average molecular weight of the polymer is between about 50 and about 1000 Daltons.

The linker L may be a hydrolytically stable linker X as described above. In other embodiments, the linker L may be linker that is unstable under physiological conditions or lung conditions such that the drug D can be released from the drug-polymer conjugate into the lung. For example, the linker X can comprise hydrolytically unstable linkers including, but not limited to, ester, thioester, and amides. It will be understood that the linker L may be chosen to be stable or unstable as desired for a particular application. The drug D may be as described above. In one example, the drug D can comprise a drug having a molecular weight of less than about 1500 or less than about 1000. It will be understood that the drug D can be a drug in its active form or a prodrug. It will be further understood that the drug D will be a drug in which pulmonary administration is desirable.

In accordance with further embodiments of the present invention, methods of controlling the rate of systemic absorption of a drug (also referred to herein as “small molecule”) pulmonarily administered are provided. The methods comprise covalently attaching to the drug a hydrophilic polymer molecule to form a drug-polymer conjugate. As noted above, the attachment of the hydrophilic polymer can have the effect of increasing the ability of an otherwise non-absorbed drug to be absorbed. Further, the rate of systemic absorption of the drug can be decreased by increasing the size of the attached hydrophilic polymer. Without wishing to be bound by theory, it is believed that increasing the size of the hydrophilic polymer slows the crossing of the conjugate through the pulmonary epithelium.

In some embodiments, the half-life of elimination of the drug-conjugate from the lung will be from about 0.5 hours to about 12 hours, or from about 1 to about 10 hours, or from about 1.5 to about 8 hours, or from about 2 to about 6 hours, or from about 2.5 to about 4 hours. In some embodiments, the half-life of elimination is less than about 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 hours. In one or more embodiments, the rate of crossing, K, is less than about 0.057, 0.060, 0.063, 0.066, 0.069, 0.073, 0.077, 0.082, 0.087, 0.092, 0.099, 0.107, 0.117, 0.126, 0.139, 0.154, 0.173, 0.462, 0.693, or 1.39 per hour. Generally, the half-life of elimination from the lung of pulmonary administered drug-polymer conjugates according to the invention can be predicted by the following equation: t_(1/2)−el=12.84*(1−e^(−kMW)), where k=0.000357, MW=molecular weight in Daltons, and t_(1/2)=elimination half-life in hours.

In some embodiments, the half-life of elimination from the lung of the unconjugated drug is less than about 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 minute. In some embodiments, the drug-polymer conjugate according to the invention, has a half-life of elimination from the lung that is 1.5-fold or more greater than the half-life of elimination from the lung of the unconjugated drug. For example, the half-life of elimination from the lung may be increased by 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or even 1000-fold.

In some embodiments, the attachment of the hydrophilic polymer can have the effect of causing the drug-polymer conjugate to have a lower log P than the parent unconjugated drug (as measured in the shake-flask method). In some embodiments, the log P of the drug-polymer conjugate will be lower than about 2.5, 2.0, 1.5, 1.0, 0.5, 0, −0.1, −0.5, −1.0, −1.5, −2.0, −2.5, −3.0, −3.5, or −4.0. The difference in log P between the conjugated drug and the unconjugated drug may range from about 0.2 to about 1.5, such as about 0.5 to about 1.2, about 0.4 to about 1, or about 0.5 to about 0.8.

The drug-polymer conjugate may be as described above in reference to the conjugate having a structure of P-L-D. In one example, the hydrophilic polymer is a polydisperse PEG. In one example, the hydrophilic polymer has a weight average molecular weight of between about 50 to about 4000. In another example, the hydrophilic polymer has a weight average molecular weight of between about 1000 to about 3500 or about 50 to about 1350. In yet another example, the drug-polymer conjugate exhibits a net hydrophilic character.

In accordance with further embodiments of the present invention, methods of controlling the lung residence time of a drug pulmonarily administered are provided. The methods comprise covalently attaching to the drug a hydrophilic polymer molecule to form a drug-polymer conjugate and delivering the drug-polymer conjugate by pulmonary administration. The drug-polymer conjugate may be as described above in reference to the conjugate having a structure of P-L-D. In one example, the hydrophilic polymer is a polydisperse PEG. In one example, the hydrophilic polymer has a weight average molecular weight of between about 50 and about 4000 Daltons. In another example, the hydrophilic polymer has a weight average molecular weight of between about 1000 and about 3500 Daltons or about 50 and about 1350 Daltons. In yet another example, the drug-polymer conjugate exhibits a net hydrophilic character. In some embodiments, by attaching the polymer to the drug, the residence time of the drug in the lung is increased, as compared to the residence time of a free form of the drug.

In accordance with some embodiments of the present invention, pharmaceutical compounds for pulmonary administration are provided. The pharmaceutical compound comprises a drug covalently attached to a hydrophilic polymer. The pharmaceutical compound may have a net hydrophilic character. The pharmaceutical compound can comprise the drug-polymer conjugates P-L-D described herein. In one example, the weight average molecular weight of the pharmaceutical compound is from about 50 to about 3500 Daltons.

In some embodiments, a composition comprising the pharmaceutical compound and at least one pharmaceutically acceptable excipient is provided. The at least one of the pharmaceutically acceptable excipients can be selected from the pharmaceutically acceptable excipients described herein. It will be understood that the composition can be provided in any form suitable for pulmonary administration. For example, the composition can be provided in liquid or dry form, including dry powder form. In one example, the composition can be provided as an aerosol. In one embodiment, the composition can be a spray-dried composition. In one example, the composition can comprise particles having a mass median aerodynamic diameter (MMAD) of less than about 10 microns or less than about 5 microns. The composition having an MMAD of less than about 10 microns or less than about 5 microns can comprise an aerosolized composition. Additionally, the composition can be provided in any suitable delivery device. For example, the composition can be provided in an inhaler device.

The composition may be used to treat any condition that can be remedied or prevented by administration of the particular pharmaceutical compound. Those of ordinary skill in the art appreciate which conditions a specific pharmaceutical compound can effectively treat. The actual dose to be administered will vary depend upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature.

The unit dosage of any given pharmaceutical composition be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition can be halted. In one embodiment, the drug-polymer conjugate can be provided in a unit dosage form. For example, the drug-polymer conjugate can be provided in a unit dosage form that is delivered by an inhaler device.

EXAMPLES

It is to be understood that while the invention has been described in conjunction with certain preferred and/or specific embodiments, the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples are commercially available unless otherwise indicated. The preparation of illustrative unimolecular PEG-mers is described in Example 9. All oligo(ethylene glycol) methyl ethers employed in the Examples below were monodisperse and chromatographically pure, as determined by reverse phase chromatography.

All ¹H NMR (nuclear magnetic resonance) data was generated by a 300 MHz NMR spectrometer manufactured by Bruker. A list of certain compounds as well as the source of the compounds is provided below.

2-Bromoethyl methyl ether, 92%, Aldrich;

1-Bromo-2-(2-methoxyethoxy)ethane, 90%, Aldrich;

CH₃(OCH₂CH₂)₃Br was prepared from CH₃(OCH₂CH₂)₃OH;

Tri(ethylene glycol) monomethyl ether, 95%, Aldrich;

Di(ethylene glycol), 99%, Aldrich;

Tri(ethylene glycol), 99%, Aldrich;

Tetra(ethylene glycol), 99%, Aldrich;

Penta(ethylene glycol), 98%, Aldrich;

Hexa(ethylene glycol), 97%, Aldrich;

Sodium hydride, 95% dry powder, Aldrich;

Methansulfonyl chloride, 99%, ACE;

Tetrabutyl ammonium bromide, Sigma

Example 1 Synthesis of CH₃(OCH₂CH₂)₃—NH-13-cis-Retinamide (PEG₃-13-cis-RA)

PEG₃-13-cis-RA was prepared. The overview of the synthesis is provided below.

0.1085 grams of CH₃(OCH₂CH₂)₃—NH₂ (0.6656 mmoles), 0.044 grams of 1-hydroxybenzyltriazole (“HOBT,” 0.3328 mmoles), and 0.200 g of 13-cis-retinoic acid (“13-cis-RA,” 0.6656 mmoles) were dissolved in 10 mL of benzene. To this solution was added 0.192 grams of 1,3-dicyclohexylcarbodiimide (“DCC,” 0.9318 mmoles) and the reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered and the solvent was removed using rotary evaporation. The crude product was further dried under vacuum, dissolved in 20 mL of dichloromethane, and the organic phase was washed twice with 15 mL of deionized water. The organic phase was dried over Na₂SO₄, filtered, and the solvent removed by rotary evaporation. To the recovered product was added 2 drops of dichloromethane containing 50 ppm butylated hydroxytoluene and the product was dried under vacuum. Yield 0.335 g. ¹H NMR (DMSO): δ 1.02 (singlet, 2 CH₃), 1.67 (singlet, CH₃), 3.5 (broad multiplet, PEG), 6.20 (m, 3H).

Example 2 Synthesis of CH₃—(OCH₂CH₂)₇—NH-13-cis-Retinamide (PEG₇-13-cis-RA)

0.2257 grams of CH₃(OCH₂CH₂)₇—NH₂ (0.6656 mmoles), 0.044 grams of 1-hydroxybenzyltriazole (0.3328 mmoles), and 0.200 grams of 13-cis-retinoic acid (0.6656 mmoles) were dissolved in 10 mL of benzene. To this solution was added 0.192 g 1,3-dicyclohexylcarbodiimide (0.9318 mmoles) and the resulting reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered, the solvent removed using rotary evaporation, and the product dried under vacuum. The product was dissolved in 20 mL dichloromethane and the solution was washed twice with 15 mL deionized water. The organic phase was dried over Na₂SO₄, filtered, and the solvent removed using rotary evaporation. To the recovered product was added 2 drops of dichloromethane containing 50 ppm butylated hydroxytoluene, and the product was dried under vacuum. Yield 0.426 g. ¹H NMR (DMSO): δ 1.01 (s, 2 CH₃), 1.68 (s, CH₃), 3.5 (br m, PEG), 6.20 (m, 3H).

CH₃—(OCH₂CH₂)₅—NH-13-cis-retinamide (“PEG₅-13-cis-RA”) was similarly prepared using this procedure except that CH₃(OCH₂CH₂)₅—NH₂ (“mPEG₅-NH₂”) was used in place of CH₃(OCH₂CH₂)₇—NH₂.

Example 3 Synthesis of CH₃(OCH₂CH₂)₁₁—NH-13-cis-Retinamide (PEG₁₁-13-cis-RA)

0.349 grams of CH₃(OCH₂CH₂)₁₁—NH₂ (0.6789 mmoles), 0.044 grams of 1-hydroxybenzyltriazole (0.3328 mmoles), and 0.204 grams of 13-cis-retinoic acid (0.6789 mmoles) was dissolved in 10 mL of benzene. To this solution was added 0.192 g 1,3-dicyclohexylcarbodiimide (0.9318 mmoles) and the reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered and the solvent distilled off using rotary evaporation. The product was dried under vacuum and dissolved in 20 mL dichloromethane. The solution was washed twice with 15 mL of deionized water and the organic phase dried over Na₂SO₄. The solution was filtered and the solvent was distilled off by rotary evaporation. To the recovered product was added 2 drops of dichloromethane containing 50 ppm butylated hydroxytoluene, and the product was dried under vacuum. Yield 0.541 g. ¹H NMR (DMSO): δ 1.01 (s, 2 CH₃), 1.68 (s, CH₃), 3.5 (br m, PEG), 6.20 (m, 3H).

Example 4 Synthesis of PEG-3-Naloxol

The structure of the naloxol, an exemplary small molecule drug, is shown below.

This molecule was prepared (having a protected hydroxyl group) as part of a larger synthetic scheme as described in Example 5.

Example 5 Synthesis of α,β-6-CH₃—(OCH₂CH₂)₁-Naloxol (α,β-PEG₁-Nal)

α,β-PEG₁-naloxol was prepared. The overview of the synthesis is provided below.

5.A. Synthesis of 3-MEM-Naloxone

Diisopropylethylamine (390 mg, 3.0 mmole) was added to a solution of naloxone.HCl.2H₂O (200 mg, 0.50 mmole) in CH₂Cl₂ (10 mL) with stirring. Methoxyethyl chloride (“MEMCl,” 250 mg, 2.0 mmole) was then added dropwise to the above solution. The solution was stirred at room temperature under N₂ overnight.

The crude product was analyzed by HPLC, which indicated that 3-MEM-O-naloxone (1) was formed in 97% yield. Solvents were removed by rotary evaporation to yield a sticky oil.

5.B. Synthesis of α and β Epimer Mixture of 3-MEM-Naloxol (2)

3 mL of 0.2 N NaOH was added to a solution of 3-MEM-naloxone (1) (obtained from 5.A. above, and used without further purification) in 5 mL of ethanol. To this was added a solution of NaBH₄ (76 mg, 2.0 mmole) in water (1 mL) dropwise. The resulting solution was stirred at room temperature for 5 hours. The ethanol was removed by rotary evaporation followed by addition of a solution of 0.1 N HCl solution to destroy excess NaBH₄ and adjust the pH to a value of 1. The solution was washed with CHCl₃ to remove excess methoxyethyl chloride and its derivatives (3×50 mL), followed by addition of K₂CO₃ to raise the pH of the solution to 8.0. The product was then extracted with CHCl₃ (3×50 mL) and dried over Na₂SO₄. The solvent was removed by evaporation to yield a colorless sticky solid (192 mg, 0.46 mmole, 92% isolated yield based on naloxone.HCl.2H₂O).

HPLC indicated that the product was an α and β epimer mixture of 3-MEM-naloxol (2).

5.C. Synthesis of α and β Epimer Mixture of 6-CH₃—OCH₂CH₂—O-3-MEM-Naloxol (3a)

NaH (60% in mineral oil, 55 mg, 1.38 mmole) was added into a solution of 6-hydroxyl-3-MEM-naloxol (2) (192 mg, 0.46 mmole) in dimethylformamide (“DMF,” 6 mL). The mixture was stirred at room temperature under N₂ for 15 minutes, followed by addition of 2-bromoethyl methyl ether (320 mg, 2.30 mmole) in DMF (1 mL). The solution was then stirred at room temperature under N₂ for 3 hours.

HPLC analysis revealed formation of a mixture of α- and β-6-CH₃—OCH₂CH₂—O-3-MEM-naloxol (3) in about 88% yield. DMF was removed by a rotary evaporation to yield a sticky white solid. The product was used for subsequent transformation without further purification.

5.D. Synthesis of α and β Epimer Mixture of 6-CH₃—OCH₂CH₂-naloxol (4)

Crude α- and β-6-CH₃—OCH₂CH₂—O-3-MEM-naloxol (3) was dissolved in 5 mL of CH₂Cl₂ to form a cloudy solution, to which was added 5 mL of trifluoroacetic acid (“TFA”). The resultant solution was stirred at room temperature for 4 hours. The reaction was determined to be complete based upon HPLC assay. CH₂Cl₂ was removed by a rotary evaporator, followed by addition of 10 mL of water. To this solution was added sufficient K₂CO₃ to destroy excess TFA and to adjust the pH to 8. The solution was then extracted with CHCl₃ (3×50 mL), and the extracts were combined and further extracted with 0.1 N HCl solution (3×50 mL). The pH of the recovered water phase was adjusted to a pH of 8 by addition of K₂CO₃, followed by further extraction with CHCl₃ (3×50 mL). The combined organic layer was then dried with Na₂SO₄. The solvents were removed to yield a colorless sticky solid.

The solid was purified by passage two times through a silica gel column (2 cm×30 cm) using CHCl₃/CH₃OH (30:1) as the eluent to yield a sticky solid. The purified product was determined by ¹H NMR to be a mixture of α- and β epimers of 6-CH₃—OCH₂CH₂-naloxol (4) containing ca. 30% a epimer and ca. 70% 13 epimer [100 mg, 0.26 mmole, 56% isolated yield based on 6-hydroxyl-3-MEM-naloxol (2)].

¹H NMR (δ, ppm, CDCl₃): 6.50-6.73 (2H, multiplet, aromatic proton of naloxol), 5.78 (1H, multiplet, olefinic proton of naloxone), 5.17 (2H, multiplet, olefinic protons of naloxol), 4.73 (1H, doublet, C₅ proton of α naloxol), 4.57 (1H, doublet, C₅ proton of β naloxol), 3.91 (1H, multiplet, C₆ proton of α naloxol), 3.51-3.75 (4H, multiplet, PEG), 3.39 (3H, singlet, methoxy protons of PEG, a epimer), 3.36 (3H, singlet, methoxy protons of PEG, β epimer), 3.23 (1H, multiplet, C₆ proton of β naloxol), 1.46-3.22 (14H, multiplet, protons of naloxol).

Example 6 Synthesis of 6-CH₃—(OCH₂CH₂)₃-naloxol (α,β-PEG₃-Nal)

6.A. Synthesis of an α and β Epimer Mixture of 6-CH₃—(OCH₂CH₂)₃—O-3-MEM-naloxol

NaH (60% in mineral oil, 38 mg, 0.94 mmole) was added to a solution of 3-MEM-naloxol [98 mg, 0.24 mmole, from Example 5 and shown as (2) in the schematic therein] in dimethylformamide (“DMF,” 8 mL). The solution was stirred at room temperature under an atmosphere of N₂ for 15 minutes, to which was added a solution of CH₃—(OCH₂CH₂)₃Br (320 mg, 1.41 mmole) in DMF (1 mL). The resulting solution was then heated under N₂ in an oil bath for 2 hours.

HPLC analysis revealed that the desired product, a mixture of α- and β-6-CH₃—(OCH₂CH₂)₃—O-3-MEM-naloxol was formed in approximately 95% yield. DMF was removed by a rotary evaporation to yield a sticky white solid. The crude product was used without further purification.

6.B. Synthesis of α and β Epimer Mixture of 6-CH₃—(OCH₂CH₂)₃—O-naloxol (α,β-PEG₃-Nal)

The crude α- and β-6-CH₃—(OCH₂CH₂)₃—O-3-MEM-naloxol mixture from 6.A. above was dissolved in 3 mL of CH₂Cl₂ to form a cloudy solution, to which was added 4 mL of trifluoroacetic acid (“TFA”). The resulting solution was stirred at room temperature for 4 hours. HPLC analysis showed that the reaction was complete. The solvent, CH₂Cl₂, was removed by a rotary evaporation. To the remaining solution was added 5 mL of water, followed by addition of K₂CO₃ to destroy excess TFA and adjust the pH to 8. The solution was then extracted with CHCl₃ (3×50 mL). The CHCl₃ extracts were combined and extracted with 0.1 N HCl solution (3×50 mL). The remaining water phase was again adjusted to a pH of 8 by addition of K₂CO₃, followed by extraction with CHCl₃ (3×50 mL). The combined organic extracts were then dried over Na₂SO₄. Following removal of the solvents, a colorless sticky solid was obtained.

The solid was purified by passage through a silica gel column (2 cm×30 cm) twice using CHCl₃/CH₃OH (30:1) as the eluent. The purified product, a mixture of the α and β epimers of 6-CH₃—(OCH₂CH₂)₃—O-naloxol containing about equal amounts of the α and β epimers, was characterized by NMR. (46 mg, 0.097 mmole, 41% isolated yield based on 6-hydroxyl-3-MEM-O-naloxone). ¹H NMR (δ, ppm, CDCl₃): 6.49-6.72 (2H, multiplet, aromatic proton of naloxol), 5.79 (1H, multiplet, olefinic proton of naloxol), 5.17 (2H, multiplet, olefinic protons of naloxol), 4.71 (1H, doublet, C₅ proton of a naloxol), 4.52 (1H, doublet, C₅ proton of naloxol), 3.89 (1H, multiplet, C₆ proton of a naloxol), 3.56-3.80 (12H, multiplet, PEG), 3.39 (3H, singlet, methoxy protons of PEG, a epimer), 3.38 (3H, singlet, methoxy protons of PEG, β epimer), 3.22 (1H, multiplet, C₆ proton of β naloxol), 1.14-3.12 (14H, multiplet, protons of naloxol).

6.C. Separation of α-6-CH₃—(OCH₂CH₂)₃—O-naloxol and β-6-CH₃—(OCH₂CH₂)₃—O-naloxol

About 80 mg of a crude mixture of α and β epimers of PEG₃-Nal was dissolved in a minimum of CHCl₃ and loaded onto a silica gel column (2 cm×30 cm) prepared using CHCl₃. The column was carefully eluted with a CHCl₃/CH₃OH mixture (60:1). Pure α-PEG₃-Nal was the first-eluting species (26 mg, 33% isolated yield), followed by pure β-PEG₃-Nal (30 mg, 38% isolated yield). Both compounds were colorless sticky solids. α-PEG₃-Nal, ¹H NMR (δ, ppm, CDCl₃): 6.49-6.73 (2H, two doublet, aromatic proton of naloxol), 5.79 (1H, multiplet, olefinic proton of naloxol), 5.17 (2H, triplet, olefinic protons of naloxol), 4.71 (1H, doublet, C₅ proton of naloxol), 3.81 (1H, multiplet, C₆ proton of naloxol), 3.57-3.80 (12H, multiplet, PEG), 3.40 (3H, singlet, methoxy protons of PEG), 1.13-3.12 (14H, multiplet, protons of naloxone). β-PEG₃-Nal, ¹H NMR (δ, ppm, CDCl₃): 6.54-6.72 (2H, two doublet, aromatic proton of naloxol), 5.77 (1H, multiplet, olefinic proton of naloxol), 5.15 (2H, triplet, olefinic protons of naloxol), 4.51 (1H, doublet, C₅ proton of naloxol), 3.58-3.78 (12H, multiplet, PEG), 3.39 (3H, singlet, methoxy protons of PEG), 3.20 (1H, multiplet, C₆ proton of naloxol), 1.30-3.12 (13H, multiplet, protons of naloxol).

α,β-6-CH₃—(OCH₂CH₂)₅—O-naloxol (“α,β-PEG₅-Nal”) and α,β-6-CH₃—(OCH₂CH₂)₇—O—naloxol (“α,β-PEG₇-Nal”) were similarly prepared, and their individual isomers separated and isolated.

Example 7 Oral Bioavailability of PEG-Mers of Cis-Retinoic Acid and Naloxol

Female Sprague Dawley® rats (150-200 g) were obtained from Harlan Labs. They were cannulated in the external jugular vein and allowed at least 72 hours of acclimatization before the start of the study. The animals were fasted overnight (day−1), but water was provided ad libitum.

On the morning of dosing (day 0), each rat was weighed and the cannulas flushed with heparin (1000 U/mL). With the aid of a feeding tube, the animals were then dosed orally (gavage) with aqueous formulations containing either the PEGylated or the free drug. The dose was determined on a mg/kg body weight basis. The total volume of the dose did not exceed 10 mL/kg. At specific time intervals (1, 2 and 4 hours), blood samples (approximately 1.0 mL) were removed through the cannula, placed in 1.5 mL centrifuge tubes containing 14 μL of heparin, mixed and centrifuged to separate the plasma. The plasma samples were frozen (<−70° C.) until assayed. The plasma samples were purified by a precipitation technique and the analyte extracted and assayed using a high performance liquid chromatography (LC) method with a mass selective detector (MSD). Standard samples were prepared in the same way to create a standard curve, from which the concentration of unknown samples could be extrapolated (see results in Table II). When appropriate, an internal standard was used in the analysis.

Selected properties of the tested compounds (such as the molecular weight and solubility) are summarized in Table I. The in-vitro enzyme binding activity of some of the tested compounds are also reported as IC₅₀ values in Table 1

TABLE 1 Selected Properties of Tested Compounds Molecular IC₅₀ Drug Weight Solubility (μM) (nM)* 13-cis-Retinoic Acid 300.45 0.47 — (parent drug) PEG₃-13-cis-RA 445.64 3.13 — PEG₅-13-cis-RA 549.45 soluble — PEG₇-13-cis-RA 621.45 58.3 — PEG₁₁-13-cis-RA 797.45 soluble — Naloxone “Nal” (parent drug) 327.37 soluble as HCl salt 6.8 α isomer of PEG₃-Nal 475.6 soluble 7.3 β isomer of PEG₃-Nal 475.6 soluble 31.7 α isomer of PEG₅-Nal 563.0 soluble 31.5 β isomer of PEG₅-Nal 563.0 soluble 43.3 α isomer of PEG₇-Nal 652.0 soluble 40.6 β isomer of PEG₇-Nal 652.0 soluble 93.9 α isomer of PEG₉-Nal 740.0 soluble 64.4 β isomer of PEG₉-Nal 740.0 soluble 205.0 Hydroxyzine “Hyd” 374.91 soluble as HCl salt 48.8 (parent drug) PEG₁-Hyd 433.0 soluble 70.3 PEG₃-Hyd 521.0 soluble 105.0 PEG₅-Hyd 609.0 soluble 76.7 Cetirizine “Cet” (parent drug) 388.89 soluble as HCl salt 77.1 PEG₁-Cet 446.0 soluble 61.0 PEG₃-Cet 534.0 soluble 86.4 PEG₅-Cet 622.0 soluble 128.0 *Mu-opiate binding activity for naloxone series of compounds Histamine H-1 binding activity for hydroxyzine and cetirizine series of compounds

The oral bioavailabilities of the retinoic acid series of compounds were calculated and the results provided in Table II. All the data was normalized to a 6 mg/kg dose. The plasma concentration versus time profiles for these compounds are provided in FIG. 1.

TABLE II Oral Bioavailabilities of the Retinoic Acid Series of Compounds Mean Plasma Concentration (ng/mL) ± SD N Drug 1 hr 2 hr (rats) 13-cis-Retinoic Acid 43.3 ± 24.0 23.3 ± 14.8 3 PEG₃-13-cis-RA 131.8 ± 55.0  158.0 ± 133.0 7 PEG₅-13-cis-RA 77.7 ± 31.6 61.6 ± 57.1 4 PEG₇-13-cis-RA 44.0 ± 13.0 38.7 ± 4.2  3 PEG₁₁-13-cis-RA 21.8 ± 7.1  58.2 ± 43.5 4

The oral bioavailability of each isomer in the naloxone series of compounds was calculated and is provided in Table III. The oral naloxone dose was either 5 or 10 mg/kg and the doses for the PEGylated compounds were normalized to 1 mg/kg dose. The plasma concentration versus time profiles for these compounds is provided in FIG. 2.

TABLE III Oral Bioavailabilities of the Naloxone Series of Compounds Mean Plasma Concentration (ng/mL) ± SD N Drug 1 hr 2 hr (rats) Naloxone 3.67 ± 1.05 3.11 ± 0.46 4 α-PEG₃-Nal 37.28 ± 4.99  14.92 ± 5.27  5 β-PEG₃-Nal 53.79 ± 5.19  22.47 ± 8.78  5 α-PEG₅-Nal 27.37 ± 10.82 15.38 ± 6.65  6 β-PEG₅-Nal 69.34 ± 15.03 36.92 ± 15.84 5 α-PEG₇-Nal 40.08 ± 16.61 39.51 ± 9.57  4 β-PEG₇-Nal 50.41 ± 36.44 50.08 ± 25.28 4

The above results show that PEGylation of small, lipophilic compounds like retinoic acid and naloxone (the free base form) increases their solubility and oral bioavailability. On the other hand, attachment of oligomeric PEGs also increases the molecular weight of the parent compound (greater than about 500 Daltons), which in turn can restrict the oral permeation of highly water soluble compounds, particularly with increasing PEG-mer length, as seen for example with PEG₇-13-cis-RA and PEG₁₁-13-cis-RA.

Example 8 Transport Across the Blood Brain Barrier (BBB) of Peg-Mers of Cis-Retinoic Acid and Naloxone,

As utilized for these experiments, the in situ brain perfusion technique employed the intact rat brain to (i) determine drug permeation across the BBB under normal physiological conditions, and (ii) to study transport mechanisms such as passive diffusion verses carrier mediated transport.

Perfusion was performed using the single time-point method. Briefly, the perfusion fluid (perfusate) containing the test compound(s) was infused into rats via the left external carotid artery at a constant rate by an infusion pump (20 mL/min). Perfusion flow rate was set to completely take over fluid flow to the brain at normal physiologic pressure (80-120 mm Hg). The duration of the perfusion was 30 seconds. Immediately following the perfusion, the brain vasculature was perfused for an additional 30 seconds with drug-free perfusate to remove residual drug. The pump was turned off and the brain was then immediately removed from the skull. Left-brain samples from each rat were first weighed and then homogenized using a Polytron homogenizer. Four (4) mL of 20% methanol was added to each rat brain for homogenization. After homogenization, the total volume of homogenate was measured and recorded.

A measured amount of the homogenate was diluted with organic solvent and subsequently centrifuged. The supernatant was removed, evaporated in a stream of nitrogen and reconstituted and analyzed by LC/MS/MS. Quantification of drug concentrations in brain homogenate was performed against calibration curves generated by spiking the drugs into blank (i.e. drug-free) brain homogenate. Analysis of the drug concentrations in brain homogenates was carried out in triplicate, and the values were used to calculate the brain uptake rate in pmole per gram of rat brain per second of perfusion.

Each perfusion solution contained atenolol (target concentration, 50 μM), antipyrine (target concentration, 5 μM) and a test compound (13-cis-retinoic acid, PEG_(n)-13-cis-retinoic acid, naloxone or PEG_(n)-Nal) at a target concentration of 20 μM.

The BBB uptake of each compound tested was calculated, normalized and recorded in Table IV. All the data was normalized to a 5 μM dosing solution at 20 mL/min perfusion rate for 30 sec.

TABLE IV Blood-Brain Barrier (BBB) Uptake for Tested Compounds Normalized Brain Uptake Rate in pmole/gm brain/sec N Drug (Mean ± SD) (rats) Atenolol (low standard) 0.7 ± 0.9 4 Antipyrine (high standard) 17.4 ± 5.7  4 13-cis-Retinoic Acid 102.54 ± 37.31  4 PEG₃-13-cis-RA 79.65 ± 20.91 4 PEG₅-13-cis-RA 58.49 ± 13.44 3 PEG₇-13-cis-RA 24.15 ± 1.49  3 PEG₁₁-13-cis-RA 17.77 ± 1.68  3 Naloxone 15.64 ± 3.54  3 PEG₃-Nal 4.67 ± 3.57 3 PEG₅-Nal 0.96 ± 0.36 3 PEG₇-Nal (α isomer) 0.94 ± 0.32 3 PEG₇-Nal (β isomer) 0.70 ± 0.19 3 Hydroxyzine 355.89 ± 59.02  3 PEG₅-Hyd 131.60 ± 15.84  3 PEG₇-Hyd 12.01 ± 2.97  3 Cetrizine 1.37 ± 0.37 3 PEG₅-Cet 4.32 ± 0.26 3 PEG₇-Cet 1.13 ± 0.05 3

The above results demonstrate that PEGylation of a lipophilic compound such as 13-cis-retinoic acid can significantly reduce its brain uptake rate (“BUR”), e.g., by a factor of four in the case of PEG₇-13-cis-RA, and by a factor of five in the case of PEG₁₁-13-cis-RA as compared to the parent compound “13-cis-retinoic acid”. In the case of naloxone, a reduction in BUR of 16 times was observed for PEG₅-Nal and PEG₇-Nal. With respect to hydroxyzine, the BUR was reduced about 29 times when administered as PEG₇-Hyd. The relatively minimal transport of cetirizine across the blood-brain barrier was not altered significantly when administered as PEG₇-Cet.

Thus, overall, it was surprisingly discovered that by attaching small water-soluble polymers to small molecule drugs such as these, one can optimize a drug's delivery profile by modifying its ability to cross biological membranes, such as the membranes associated with the gastro-intestinal barrier, the blood-brain barrier, the placental barrier, and the like. More importantly, it was discovered that, in the case of orally administered drugs, attachment of one or more small water-soluble polymers is effective to significantly reduce the rate of transport of such drugs across a biological barrier such as the blood-brain barrier. Ideally, the transport of such modified drugs through the gastro-intestinal tract is not adversely impacted to a significant degree, such that while transport across the biological barrier such as the blood-brain barrier is significantly impeded, the oral bioavailability of the modified drug is retained at a clinically effective level.

The data generated in Examples 7 and 8 was plotted in order to compare the effect of PEG size on the relative oral bioavailability and BBB transport of 13-cis-retinoic acid and naloxone, respectively. See FIGS. 3-7. In FIG. 3, the effect of attaching each of a PEG 3-mer, a PEG 5-mer, a PEG 7-mer and a PEG 11-mer to 13-cis-retinoic acid on its oral bioavailability is examined. In FIG. 4, the effect of covalent attachment of these various PEG-mers on the blood-brain barrier transport of 13-cis-retinoic acid is examined. In FIG. 5, the effect of covalent attachment of each a PEG 3-mer, PEG 5-mer and a PEG 7-mer on the oral bioavailability of naloxone is examined. FIG. 6 demonstrates the effect of covalent attachment of such PEG-mers on the blood brain-barrier transport of naloxone. FIG. 7 shows that the PEG_(n)-Nal compounds had a higher oral bioavailability than naloxone. As can be seen from these figures, as the size of the PEG oligomer increases, the BBB uptake rate significantly decreases, while the oral bioavailability increases relative to that of the parent molecule.

The difference in oral bioavailability between the α- and β-isomers of naloxone may be due to the differences in their physicochemical properties. One isomer appears to be slightly more lipophilic than the other isomer, and thereby results in a small difference in oral bioavailability.

Example 9 In-Vitro Metabolism of PEG-Naloxol

An in vitro method was developed to study the effect of PEGylation on the Phase H metabolism (glucuronidation) of naloxone. The procedure calls for the preparation of a NADPH regenerating system (NRS) solution. The NRS solution is prepared by dissolving sodium bicarboante (22.5 mg) in 1 mL of deionized water. Into this solution B-nicotinamide adenine dinucleotide phosphate sodium salt or NADP (1.6 mg), glucose-6-phosphate (7.85 mg), glucose-6-phosphate dehydrogenase (3 μL), uridine 5-diphosphoglucuronic acid trisodium salt or UDPGA (2.17 mg), adenosine 3′-phosphate 5′-phosphosulfate lithium salt or PAPS (0.52 mg), and 1 M magnesium chloride solution (10 μL) were added. After the solids were all dissolved, the solution was stored in an ice bath.

30 mM test article stock solutions were prepared by dissolving weighed amounts of naloxone HCl, 6-mPEG₃-O-Naloxone, α-6-mPEG₅-O-naloxone, and α-mPEG₇-O-Naloxone in 1 mL of deionized water.

Male Sprague Dawley rat microsomes (0.5 mL at 20 mg/mL concentration; M00001 from In-vitro Technologies, Baltimore, Md.) were removed from the freezer and thawed in an ice bath. Forty μL of the liver microsomes were diluted to 100 μL with 60 μL of deionized water in a test vial. To the test vial, tris buffer, pH 7.4 (640 μL) and a test article stock (10 μL) were added to have 750 μL volume.

Each test vial and the NRS solution were separately placed in a 37° C. water bath for 5 minutes. The NRS solution (250 μL) was added into each test vial. The reaction timer was started at the addition of the NRS to the first test vial. Each sample (200 μL) was collected and then perchloric acid (20 μL) was added to terminate the reaction. The samples were collected at the following time points: 0-2, 20, 40 and 60 minutes. All of the terminated test vials were stored in an ice bath.

Acetonitrile (100 μL) was added into each test vial, which was then centrifuged at 3000×g for 5 minutes. Supernatant (230 μL) was withdrawn and then 10 μL of the test solution was assayed by an LC/MS method. The concentration of test article in each sample was measured and recorded at each time point.

Table V lists the percentage of active remaining after incubation with liver microsomes.

TABLE V Percentage of Active Remaining After Incubation with Liver Microsomes Time α-PEG₃- β-PEG₃- α-PEG₅- α-PEG₇- (min) naloxone Nal Nal Nal Nal 0 100.0 100.0 100.0 100.0 100.0 20 47.1 64.8 83.9 84.1 87.4 40 27.6 51.7 75.2 75.6 81.6 60 15.6 45.7 69.6 69.2 76.9

In view of the results in Table V, it is possible to conclude that PEGylation with an oligomer decreases that rate of glucuronidation for a small molecule such as naloxol. Furthermore, as the PEG oligomer chain increases, the rate of glucuronidation decreases. In addition, comparison of α-isomers and β-isomers of PEG₃-naloxol, shows that the β-isomer is a poor substrate for cytochrome P450 isozymes in the isolated rat liver microsomes. This observation confirms the in-vivo data illustrated in FIG. 7.

Turning to the data in FIGS. 8 and 9, it appears that attachment of small PEGs can be effective in decreasing the rate of drug metabolism (as indicated by glucuronide formation in the case of naloxone). The higher levels of the β-isomer in the blood when compared to the α-isomer is likely due to a significant prevention of the first pass effect, that is to say, a significant prevention of the extent of first pass metabolism (FIG. 7), resulting from covalent attachment of the oligomeric PEG molecule. The PEG molecule may create steric hinderance and/or hydrophilic or hydrophobic effects, which when the PEG is attached to the β-isomer form, alters the affinity of the β-isomer conjugate to cytochrome P450 isozymes to a greater degree than when the PEG is attached to the α-isomer form. The levels of β-isomer metabolite are lower when compared to the α-isomer metabolite and unPEGylated naloxone.

Example 10 Activity of Various Opiod Antagonists on μ-Opiate Receptors

In a separate series of experiments, the bioactivity of naloxone, other opiod antagonists, and various conjugates on μ-opiate receptors was determined in-vitro. The results are summarized in Table VI.

TABLE VI Activity of Naloxone and PEG_(n)-6-Naloxol Conjugates on μ-Opiate Receptors, in-vitro. Molecular EC₅₀ Compound Weight (nM) Naloxone 327.4 6.8 3-PEG₃-O-naloxone 474 2910.0 6-NH₂-naloxone 601 29.2 PEG₅₅₀-6-NH-naloxone (PEG₁₃ amide) 951 210.0 α-6-naloxol 329 2.0 β-6-naloxol 329 10.8 α-PEG₃-Nal 475.6 7.3 β-PEG₃-Nal 475.6 31.7 α-PEG₅-Nal 563 31.5 β-PEG₅-Nal 563 43.3 α-PEG₇-Nal 652 40.6 β-PEG₇-Nal 652 93.9

In the table above, for each compound, the bioactivity is provided as a measure of the relative bioactivity of each of the various PEG conjugates in comparison to parent drug. The EC₅₀ is the concentration of agonist that provokes a response halfway between the baseline and maximum response in a standard dose-response curve. As can be seen from the above data, each of the PEG_(n)-Nal conjugates is bioactive, and in fact, all of the 6-naloxone or naloxol conjugates maintained a degree of bioactivity that is at least 5% or greater than that of the parent drug, with bioactivities ranging from about 5% to about 35% of the bioactivity of the unmodified parent compound. In terms of bioactivity, PEG₅₅₀-6-NH-naloxone possesses about 13% of the bioactivity of the parent compound (6-NH₂-naloxone), α-PEG₃-Nal possesses about 30% of the bioactivity of the parent compound (α-6-OH-naloxol), and β-PEG₃-Nal possesses about 35% of the bioactivity of the parent compound (α-6-OH-naloxol).

Example 11 Method of Making Substantially Unimolecular Weight Oligo(Ethylene Glycol) Methyl Ethers and their Derivatives

The unimolecular (monodisperse) PEGs of the present invention were prepared as set forth in detail below. These unimolecular PEGs were particularly advantageous in providing the modified active agents of the present invention, and in imparting the desired modification of barrier transport properties of the subject active agents.

The method exemplified below represents another aspect of the present invention, that is, a method for preparing monodisperse oligo(ethylene oxide) methyl ethers from low molecular weight monodisperse oligo(ethylene glycol)s using halo-derivatized (e.g., bromo derivatized) oligo(ethylene oxide). Also provided herein, in another aspect of the invention, is a method of coupling oligo(ethylene oxide) methyl ether (from a unimolecular weight composition) to an active agent using a halo-derivatized oligo(ethylene oxide) methyl ether.

Schematically, the reaction can be represented as follows:

10.A. Synthesis of CH₃O—(CH₂CH₂O)₅—H with CH₃OCH₂CH₂Br

Tetra(ethylene glycol) (55 mmol, 10.7 g) was dissolved in 100 mL of tetrahydrofuran (“THF”) and to this solution was added KOtBu (55 mL, 1.0M in THF) at room temperature. The resulting solution was stirred at room temperature for 30 minutes, followed by dropwise addition of CH₃OCH₂CH₂Br (55 mmol, 5.17 mL in 50 mL THF). The reaction was stirred at room temperature overnight, followed by extraction with H₂O (300 mL)/CH₂Cl₂ (3×300 mL). The organic extracts were combined and then dried over anhydrous Na₂SO₄. After filtering off the solid drying agent and removing the solvent by evaporation, the recovered crude residue was purified by column chromatography using a silica gel column (CH₂Cl₂: CH₃OH=60:1˜40:1) to give pure penta(ethylene glycol) monomethyl ether (yield 35%). ¹H NMR (CDCl₃) δ 3.75-3.42 (m, 20H, OCH₂CH₂O), 3.39 (s, 3H, MeO). 10.B. Synthesis CH₃O—(CH₂CH₂O)₇—H Using MeOCH₂CH₂Br

To a solution of hexa(ethylene glycol)(10 g, 35 mmole) and 2-bromoethyl methyl ether (4.9 g, 35 mmole) in THF (100 mL) was slowly added sodium hydride (2.55 g, 106 mmole). The solution was stirred at room temperature for two hours. HPLC indicated that mPEG₇-OH was formed in about 54% yield. The reaction was then stopped by the addition of diluted hydrochloride acid to destroy excess sodium hydride. All solvents were removed using a rotary evaporator to give a brown sticky liquid. Pure mPEG₇-OH was obtained as a colorless liquid (4.9 g, 41% isolated yield) by using semi-preparative HPLC (20 cm×4 cm, C18 column, acetonitrile and water as mobile phases). ¹H NMR (CDCl₃): 2.57 ppm (triplet, 1H, OH); 3.38 ppm (singlet, 3H, CH₃O); 3.62 ppm (multiplet, 30H, OCH₂CH₂).

10.C. Synthesis of CH₃O—(CH₂CH₂O)₅—Br

Triethyl amine (5.7 ml, 40 mmol) was added to CH₃O—(CH₂CH₂O)₅—OH (5.0 g, 20 mmol) with stirring. The solution was cooled in an ice bath under N₂, and 2.5 ml of methanesulfonyl chloride (32 mmol) was added dropwise over 30 minutes. The solution was then stirred overnight at room temperature. Water (40 ml) was added to the reaction mixture and the solution was extracted with CH₂Cl₂ (3×150 ml) and the organic phase was washed with 0.1 N HCl (3×80 ml) and water (2×80 ml). After drying with Na₂SO₄ and removal of solvent, a light brown liquid was obtained. The product and Bu₄NBr (12.80 g, 39.7 mmol) were dissolved in CH₃CN (50 ml), and the resulting solution was stirred under N₂ at 50° C. for 15 hours. After cooling to room temperature, CH₃CN was removed by rotary evaporation to give a red liquid, which was dissolved in 150 ml water and extracted with EtOAc (2×200 ml). The organic phase was combined, washed with water, and dried over Na₂SO₄. After the removal of solvent, a red liquid was obtained (4.83 g, 77.4%). ¹H NMR (300 Hz, CDCl₃): δ 3.82 (t, 2H), 3.67 (m, 14H), 3.51 (m, 2H), 3.40 (s, 3H).

Synthesis of mPEG3 N-Mefloquine

To a methanol solution (5 mL) of mefloquine HCl salt (200 mg, 0.48 mmol) and mPEG₃-Butyaldehyde (280 mg, 1.20 mmol) was added sodium cyanoborohydride (60 mg, 0.96 mmol) water solution (1 mL). The resulting solution was heated under nitrogen with stirring in an oil bath at 50° C. for 16 hours. HPLC showed that the reaction was complete. All solvents were then removed by a rotary evaporator to give a crude product. After purified by a preparative reverse phase HPLC, pure mPEG-3-N-Mefloquine conjugate was obtained as a colorless sticky liquid (160 mg, 0.27 mmol, 56% isolated yield). ¹H NMR (CDCl₃, ppm): 8.15 (multiplet, 3H, aromatic ring); 7.73 (triplet, 1H, aromatic ring); 5.86 (doublet, 1H, CH); 3.67 (multiplet, 14H, PEG back bone); 3.52 (singlet, 3H, PEG-OCH₃); 3.18 (multiplet, 2H, PEG-CH₂); 0.52-2.74 (multiplet, 13H, PEG and cyclohexyl protons).

Schematically, the reaction can depicted as follows:

Example 12 Preparation of PEG-FITC Molecules and Experimental Protocols

12.A—PEG-FITC Conjugates

A series of PEG-fluorescein isothiocynate (FITC) molecules were prepared whereby the different sized PEGs were covalently attached via the thiocyanate functionality on the FITC resulting in a FITC-thiourea-PEG conjugate. Use of the mPEGn-OH reagent in the conjugation reaction for PEG sizes 0.55K, 1K, 2K, 5K and 20K Daltons ensured a 1:1 ratio of PEG to FITC, i.e. each PEG-FITC conjugate consisted of one MAC molecule per PEG molecule. However, the conjugate composed of 3.4 K-PEG-FITC was generated using the HO-PEGn-OH dial reagent, which allows for the possibility of two different species, either two FITC (a “dumbbell”) or one FITC molecule per PEG. Since unconjugated FITC is highly reactive and would bind to many proteins in tissues and cells, sodium fluorescein was employed as a control, to determine the residence time of the parent non-PEGylated molecule in the lung.

PEG was conjugated with FITC. Note that in the case of PEG(OH)FITC (the 3.4K PEG-FITC species), the methyl group at the end of the PEG chain would be replaced by a hydroxyl group, or by a second FITC moiety. The structure of the PEG-FITC conjugate is shown below.

12.B—Design of Animal Experiments

To determine the effect of PEG length on residence time in the lung, a series of in vivo experiments was performed in which each PEG-FITC species was administered intratracheally (IT) to rats, and the amount remaining in the lung was determined at progressive time points. For each species, 30 μg PEG-FITC, prepared in PBS/0.5% BSA, was administered IT to rats (300 μL of a 100 μg/mL solution). Animals were sacrificed in triplicate at the following time points: predose, 10 min (0.17 h), 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 168 h (7 days), and subjected to bronchoalveolar lavage (BAL) with a single wash, using 0.9% NaCl (154 mM NaCl—physiological saline concentration). Following the lavage, lungs were excised, and samples of serum were extracted. The resulting lavage solution was then centrifuged to pellet the cellular fraction (BAL cells) and separate it from the BAL supernatant. An outline of the approach is shown in FIG. 11.

12.C—Quantitation of Test Molecules in Biological Matrices

Quantitation of PEG-FITC concentrations in the various compartments was performed using a Tecann™ Genios Pro microplate reader to detect the fluorescence intensity of the samples under conditions suitable for detection of fluorescein (λex 485 nm, λem 535 nm). Samples from BAL, BAL cells and serum were read directly in microtiter plates with no prior treatment. To quantitate PEG-FITC remaining in the residual lung material, however, the excised lungs were homogenized and PEG-FITC was extracted in ethanol.

For all samples, quantitation was performed by interpolation from a standard curve generated from known concentrations of the relevant molecule prepared in the correlating matrix. Thus for samples of BAL and BAL cells, the standard samples were prepared in 0.9% NaCl, for serum samples, the standards were prepared in commercially obtained rat serum, and for lung extracts, the standard samples were prepared in PBS:Ethanol (1:3). Lower limits of quantitation for each molecule tested in the respective matrices is shown in Table VII below.

TABLE VII Lower limits of quantitation for PEG-FITC molecules. PBS:EtOH Molecule BAL (0.9% NaCl) Serum (lung extract) Sodium fluorescein 2.5 nM   0.9 ng/mL  4 nM 1.5 ng/mL  0.24 nM 0.09 ng/mL 0.55K-PEG-FITC 5 nM  4 ng/mL ND ND  0.5 nM  0.4 ng/mL 1K PEG-FITC 5 nM  6 ng/mL ND ND 0.24 nM 0.27 ng/mL 2K PEG-FITC 5 nM 12 ng/mL 8 nM 18 ng/mL 0.24 nM 0.55 ng/mL 3.4K PEG-FITC 5 nM 18 ng/mL 8 nM 30 ng/mL   2 nM   7 ng/mL 5K PEG-FITC 5 nM 30 ng/mL 4 nM 25 ng/mL 0.24 nM  1.5 ng/mL 20K PEG-FITC 5 nM 110 ng/mL  ND ND 0.24 nM   5 ng/mL ND—not determined. 12.D—Data Analysis

Concentrations of PEG-FITC were interpolated from a standard curve, and were analyzed using GraphPad Prism 4.01 software and Microsoft Excel. For BAL studies, the total amount remaining was calculated for each animal from the product of the concentration and the BAL volume recorded. For BAL cells, the pellet was resuspended in 1 mL; the amount present was calculated accordingly. For residual lung tissue, the amount present in both lungs (μg/lung) was calculated from the product of the concentration and the homogenate volume. Note that although the nominal dose delivered in each experiment was 30 μg, the dosing solution was analyzed from the same standard curve for each experiment and the actual delivered dose was calculated. The data shown throughout this specification are thus corrected for each PEG-FITC species respectively and are represented as percentage dose administered. The doses delivered in each experiment are represented in Table VIII below.

TABLE VIII Pharmacokinetic data from in vivo experiments. BAL BAL Dose Molecule MW BAL k-el t_(1/2)-el BAL t₉₀ t₉₉ Lung t_(1/2)-el (μg) Sodium fluorescein 376 1.3253 0.523 1.74 3.5 0.5021 40 0.55K-PEG-FITC 810 0.2932 2.364 7.85 15.7 2.351 50 1K PEG-FITC 1143 0.1391 4.984 16.56 33.1 5.179 45 2K PEG-FITC 2303 0.0968 7.161 23.79 47.6 7.595 50 3.4K PEG-FITC 3655 0.0770 9.005 29.91 59.8 13.08 40 5K PEG-FITC 6149 0.0577 12.02 39.93 79.9 11.8 53 20K PEG-FITC 21930 0.0551 12.57 41.76 83.5 12.51 55

Example 13 BAL Supernatant

The concentration of PEG-FITC remaining in BAL supernatant was plotted versus time for each PEG-FITC species as shown in FIG. 12. The concentrations were determined in accordance with the protocols in Example 12, and the concentration-time relationship of each PEG-FITC species in BAL is expressed as percentage dose. Analysis of the amount of PEG-FITC remaining in the BAL supernatant at increasing time points revealed that each PEG-FITC species was eliminated from the BAL in a manner described by a single exponential curve on a concentration-time curve. As the molecule's PEG chain length increases, its lung residence time becomes more prolonged, as indicated by the increased half-life for elimination from BAL (t_(1/2)−el), as shown in FIG. 13. The elimination half life (t_(1/2)−el) determined from the concentration-time plots in FIG. 12 is plotted against molecular weight (MW), and produces an exponential association relationship, described by the equation: t_(1/2)−el=12.84*(1−e^(−kMW)), where k=0.000357, MW=molecular weight in Daltons, and t_(1/2)=elimination half-life in hours.

Conjugation to FITC of a PEG as small as even 0.55 K causes an extension in residence time, the t1/2−el increasing from <0.52 h to 2.36 h. The t1/2−el calculated for sodium fluorescein (0.52 h) is an overestimate, since its elimination from the lung was so rapid that at the first time point (10 min) less than 50% of the delivered drug was recovered. For all the PEG conjugated species, by contrast, the majority of the delivered dose was recovered in the BAL at the earliest time point. With the exception of 3.4K-PEG-FITC, over 80% of the dose administered was recovered in BAL 10 min following IT administration. In the case of 3.4K-PEG(OH)-FITC, approximately 60% of the initial dose is recovered at 10 min, and this value remains relatively unchanged at the next time point, 3 h. The reason for this is not clear, but the calculated concentration arises from readings taken from three animals, suggesting that technical error is unlikely. It is possible that the PEG(OH) reagent is more reactive with endogenous molecules than the mPEG which is conjugated to FITC in the other species, and that this alters the initial absorption through the lung. Alternatively, the presence of two FITC moieties per molecule, as per the “dumbbell” configuration may alter the absorption or the detection characteristics of this PEG-FITC species. In all cases, the PEG-FITC was completely eliminated from BAL, although the time at which this occurred increased with PEG size; that is, at later time points, PEG-FITC concentrations in the single BAL wash fell below the lower limit of quantitation, and represented an insignificant portion of the delivered dose.

A clear relationship exists between t_(1/2)−el from BAL and molecular weight (FIG. 13). The plot shows that the maximum t_(1/2)−el generated by PEG is 12.8±0.6 h, and suggests that, for controlling the rate of elimination from the lung, the most effective range of PEG size to employ extends to approximately 3.4 K, with the maximum t_(1/2)−el reached at 5K; beyond this, therefore, there is little to gain in residence time by increasing the size of PEG. Based on the elimination rates calculated from the data above estimations can be made regarding the maximum time that the molecules can be retained in the lung. The t₉₀ and t₉₉ values represented in Table VIII demonstrate the time at which 90% and 99% respectively of the dose of each species would be eliminated. For example, for 2K-PEG-FITC, 90% of the dose is eliminated from BAL after approximately 24 h (23.8 h). It appears that the maximum extension of residence time that PEG can endow on FITC molecule is in the order of 1-2 days. If so, the equation described in FIG. 13 can be used to predict what size PEG should be conjugated to a small molecule to achieve a specific residence time in the lung, and can serve to direct decisions for candidate drug molecules.

The BAL compartment derives from a single wash of the lungs, and therefore contains molecules and cells that reside on the “airside” (lumen) of the pulmonary epithelium, in addition to those that are loosely attached to cell membranes. PEGylated compounds detected in this fraction therefore represent drug molecules that would be available for action at an extracellular target in the lung, such as those that target cell surface receptors (e.g., β-agonists used in the treatment of asthma) or that target micro-organisms that infect the lung (e.g., anti-infectives). Conversely, for systemic targets, where the lung is used as a depot, PEGylation could serve as a means to slow the absorption through the lung.

Example 14 BAL Cells

The amount of PEG-FITC detected in the BAL cells fraction at each time point is displayed in FIG. 14. The percentage of dose that associates with the BAL cells' fraction is plotted against time for each PEG-FITC species. The cellular fraction of BAL constitutes primarily alveolar macrophages and contains a minor amount of epithelial cells that may have become detached in a single wash. The association of PEG-FITC molecules with this fraction most likely, therefore, represents a clearance mechanism operating by alveolar macrophages to eliminate foreign material present in the lung. Most notable among the plots in FIG. 14 is the different behavior of the smaller PEG-FITC molecules (0.55K and 1K) from that exhibited by the larger ones (2K, 3.4K, 5K, 20K). Whereas the smaller PEG-FITC molecules are fully eliminated and show a single phase of elimination, the larger PEG-FITC molecules display a biphasic response. For these molecules an initial rise is seen, followed by a decrease at 24 h; subsequently a second phase is seen with an increase in the association of PEG-FITC in BAL cells.

One possible explanation for the observed profiles is that the longer residence of the larger PEG-FITC molecules triggers a response in macrophages that recruits additional phagocytes from the systemic circulation to remove the foreign material. The smaller PEG-FITC species, by contrast, do not remain in the lung for long enough to trigger such a response. It is worth noting that no evidence of phagocytosis is presented, nor any identification of the identities of the cell types with which the PEG-FITC associates. Alternatively, it is possible that the uptake of PEG-FITC is not the result of an active phagocytosis triggered by the presence of PEG-FITC, but rather is due a non-specific pinocytotic mechanism, caused by the fluid influx from the intratracheal procedure. PEG-FITC remaining in the lung would, in this scenario, be taken up along with the fluid. The increased residence of the larger PEG-FITC molecules would underlie the differential association with macrophages exhibited by these species.

FIG. 15 demonstrates the relative uptake of the different PEG-FITC molecules on the same ordinate scale. A few interesting features appear in this comparison. First, the non-PEGylated parent molecule, sodium fluorescein, demonstrates a relatively high association with BAL cells at the earliest time point (10 min) compared with all the PEGylated species. This may indicate that a significant portion (approximately 13%) of the unmodified molecule is recognized and cleared by macrophages immediately upon delivery to the lung. The addition of PEG, even of small chain-length, appears to prevent this rapid uptake, and might contribute the ability of PEG to prolong the residence of these molecules in the epithelial lining fluid. Future studies will address whether this phenomenon is exhibited by different classes of drug molecules when conjugated to PEG.

Second, at 48 h post dose, a significant portion of the delivered dose of the larger molecules (PEG sizes 2K, 3.4K, 5K and 20K) associates with the presumed alveolar macrophages. Furthermore, for as long as 7 days (168 h) post-administration, 5-10% of the dose still remains associated with this fraction for the 5K and 20K PEG-FITC molecules. This suggests that the larger sized PEG molecules might be able to reside for extended periods in the alveolar macrophages, and may be able to protect the FITC, which is the moiety detected in the quantitative assay, from degradation. While evidence is not presented demonstrating the integrity of the FITC-PEG molecule in these cells, the differential profiles of the non-PEGylated and PEGylated species suggests that the conjugated molecule remains intact. Indeed, if the PEG had cleaved from the FITC, a rapid clearance such as that seen with the non-PEGylated molecule would be expected.

Third, a clear dependence of percentage uptake on PEG-size appears. Thus, the actual percentage of the delivered dose that remains associated with BAL cells at later time points increases with PEG chain length. This could arise from the extended residence time of these larger molecules. Thus, the longer the molecule remains in the lung, the greater the probability that it will associate with alveolar macrophages or other phagocytes which may have been recruited. The experimental design employed here, however, cannot distinguish between macrophages that associate with PEG-FITC at an earlier time point and retain it for 7 days from macrophages which may have taken up the molecule on day 7.

Example 15 Residual Lung Material

Following the BAL procedure outlined in Example 12, the remaining lungs were excised and PEG-FITC concentrations were determined from extracts of the homogenates. FIG. 16 illustrates the percentage of the dose that remains in the residual lung material plotted against time for each PEG-FITC species.

The residual lung material extracted following the BAL procedure consists of: (i) epithelial lining fluid and alveolar macrophages that were not fully extracted during the single wash of BAL, (ii) a small amount of blood that remains in the lung and (iii) the lung tissue.

The pattern of elimination of PEG-FITC from the residual lung material resembles that seen with the BAL cells' fraction. Thus for the smaller PEG sizes (0.55K, 1K) complete elimination is seen (by 12 h and 18 h respectively). However, for the larger PEG sizes (2K, 3.4K, 5K, 20K) a different profile appears, with approximately 10-15% of the dose remaining associated with the residual lung tissue at extended times. Note that for 2K and 3.4K PEG sizes, data is only available up to 48 h (2 d) post administration. For the 5K and 20K PEG sizes, data from 168 h (7 d) post dose shows a significant amount still associated with lung tissue. Since no PEG-FITC remains in the BAL at this time point, and serum concentrations lie below detectable levels, it seems likely that the majority of the material at the 168 h time point lies in the lung tissue itself, with perhaps some also associated with macrophages in the lungs.

Example 16 Serum Concentrations of PEG-FITC

Serum concentrations of PEG-FITC were analyzed as described in Example 12. FIG. 17 shows the concentration-time relationship of PEG-FITC in serum for three PEG-FITC species. For all PEG-FITC species, concentrations in serum remained so low that they were at or close to the lower limit of quantitation at all time points; these limits lie at under 1% of the administered dose. Only sodium fluorescein, 10 min post dose, produced a peak in serum that could easily be measured. Based on a rat serum volume of approximately 8 mL the peak for fluorescein would lie at approximately 2 μg, which represents less than 10% of the administered dose. By contrast, concentrations at all other time points, and for all other molecules, represent negligible proportions of the administered dose.

No analysis of whole blood was performed. Preliminary evidence suggests that the PEG-FITC molecules do not partition heavily into blood compared with serum (data not shown). Evidence in the literature indicates that PEG molecules in the size range employed here are readily cleared from serum by the kidneys. It seems likely, therefore, that PEG-FITC that is absorbed from the lungs is cleared so rapidly by the kidneys that serum concentrations remain very low. Such a scenario suggests a model of flip-flop kinetics, in which the rate of absorption from the lung into the serum is much slower than the rate of elimination from the serum.

Example 17 Mass-Balance: Combined Lung-Derived Fractions

To analyze the mass-balance at different time points, the combined amounts of material recovered in the lung-derived fractions were calculated. FIG. 18 shows the combined amounts recovered represented by stacked histograms demonstrating the relative distribution among the compartments measured at each time point.

FIGS. 18A and 18B demonstrate that for all PEG-FITC molecules, the majority of the delivered dose is recovered at 10 min post-dose. For sodium fluorescein, less than 50% of the dose given is recovered, indicating that this molecule, in the absence of conjugated PEG, is rapidly absorbed from the lung. Although the serum compartment is not represented here, the concentrations in serum were so low that the amount in serum would not constitute a significant portion of the total dose delivered, and thus would not alter these profiles.

Two features stand out among the graphs in FIGS. 18A and 18B. The first is that at later time-points, the mass balance is not achieved, —i.e., a significant portion of the delivered dose is not recovered. Serum data is so low as to preclude the serum or blood from containing the balance of the delivered dose. Instead, the most likely explanation is that the PEG-FITC has been rapidly cleared by the kidneys, as discussed previously. The second notable feature is that the smaller molecular weight PEGs (0.55K and 1K) are fully cleared from all lung-derived compartments. The larger PEGs, by contrast, are not fully cleared even by 48 h, and for the largest sizes (5K and 20K) between 25-25% of the dose remains in the combined lung-derived compartments (the BAL cells and the residual lung tissue) even 7 days post-dose.

FIGS. 19A and 19B show the amount remaining in the combined lung departments (from FIGS. 18A and 18B) as concentration-time curves for each PEG-FITC species. The curves closely resemble those in FIG. 12 representing elimination of PEG-FITC from the BAL, and the half-lives for these exponential decay curves are very close to those seen for BAL. Thus the main factor contributing to elimination from the entire lung is the rate of elimination from the BAL.

The only species for which the elimination profile differs significantly between the BAL and the combined lung fractions is the 3.4K PEG-FITC. As discussed above, this may relate to the “dumbbell” nature of the molecule which could alter its lung retention properties.

The PEG-FITC that is eliminated from the lung is most probably absorbed directly into the systemic circulation through the pulmonary epithelium. Alternative explanations for its elimination would be clearance in the lung by metabolic degradation or removal by the mucociliary escalator, leading to oral ingestion. It is highly unlikely that either of these processes would exhibit the kinetics displayed here. If absorption into the systemic circulation does indeed underlie the elimination from the BAL, then the rates of elimination should either be equal or close to the rate of absorption into the systemic circulation.

Example 18 In Vitro Studies and Log P Data for PEG-FITC Conjugates

In vitro cell-based experiments (in a Calu-3 assay) were performed for PEG-FITC conjugates. The Calu-3 cells were cultured with an apical air-interface to mimic the in vivo epithelium. The absorption data demonstrates similar rank order of permeability rates to data from in vivo studies described above. FIG. 20 illustrates the permeability rate plotted as function of molecular weight in Calu-3 studies. FIG. 21 illustrates cell-based permeability plotted versus the in vivo absorption rate. The data shows that there is a good in vivo-in vitro correlation for PEG-FITC conjugates.

The relationship between log P and PEG size for PEG-FITC conjugates was determined using a potentiometric titration method using a Sirius GLpKa instrument. FIG. 22 shows that addition of PEG causes a significant shift in log P to a more negative value. Interestingly, no specific relationship between MW and PEG and log P is observed in the molecular weight range of 0.55 to 20 KDa.

This increasing hydrophilicity by PEG conjugation, together with the in vivo absorption and in vitro permeability data, are consistent with a model of absorption in which a hydrophobic molecule, following PEGylation, moves from a predominantly transcellular route to a predominantly paracellular one. The hydrophilic nature of the PEGylated small molecule would cause it to travel preferentially via the aqueous intercellular tight junctions. As the PEG chain length increases, however, the hydrodynamic radius grows and the diffusion rate of the molecule decreases, leading to a diminished rate of permeability through the tight junctions. The effective maximum permeability rate reached at 5K PEG may represent the structural size limitation of the tight junctions, such that “unwinding” likely occurs for the molecule to enter the intercellular pore. Such an “unwinding” process might therefore represent the rate-limiting step for PEG molecules greater than 3.4K.

Example 19 Elimination of PEG and PEG-FITC from the Lung

Polydisperse PEG having a weight average molecular weight of 2 KDa was used to study elimination of PEG and PEG-FITC from the lung. The studies were performed in accordance with the procedures in Example 12. FIG. 23 shows almost identical elimination from lung for 2K PEG and 2K PEG-FITC, suggesting that PEG properties dominate those of the small molecule at this size of PEG. FIG. 24 shows that increasing the dose of 2K PEG 10-fold does not significantly alter the elimination rate from the lung. This supports the claim for passive transport of PEG across the epithelium, since the hallmark of passive transport is that permeability rates are independent of concentration (and are therefore not saturated). In vitro permeability in Calu-3 cells shows similar rates for free 2K PEG and 2K PEG-FITC, supporting the notion that PEG dominates the properties of the PEG-small molecule conjugate. The in vitro permeability is shown in Table IX below.

TABLE IX Permeability of molecules from lung (rat IT) Elimination from Cell-based lung (rat IT) Molecule Permeability (cm/s) (hr−1) Sodium Fluorescein 3.4E−07 ± 0.4E−07 1.38 Cipro HCl 8.6E−07 ± 0.1E−07 10.24 1K PEG-FITC 5.4E−08 ± 0.6E−08 0.139 1K PEG-Cipro 7.4E−08 ± 0.2E−08 0.216 2K PEG 2.3E−08 ± 0.8E−08 0.100 2K PEG-FITC 4.7E−08 ± 0.2E−08 0.097

Example 20 PEG-Cipro Studies

1 KDa and 5 KDa polydisperse PEG molecules were conjugated to ciprofloxacin (CIPRO). In vivo absorption data was collected through rat studies, in which, following IT administration to rats, whole lungs were excised and analyzed for test article concentration. The studies showed that addition of 1K PEG to CIPRO causes significant decrease in absorption across pulmonary epithelium, evident from a decrease in disappearance from the lung, as illustrated in FIG. 25, and concomitant decrease in rate of appearance in plasma, as illustrated in FIG. 26. As shown in FIG. 27, the t_(1/2) elimination was about 4 minutes with CIPRO alone and about 3.2 hours with the 1K PEG-CIPRO conjugate (FIG. 26).

This data shows that the elimination from the lung is paralleled by appearance in plasma, thus confirming the process of absorption through the pulmonary epithelium. The increased T_(max) of 1K-PEG-CIPRO reflects the slower absorption, resulting in a longer time taken to reach the C_(max). The decreased C_(max) of the 1K-PEG-CIPRO compared with CIPRO most likely arises predominantly from the decreased rate of absorption. Although the elimination from serum may be slightly decreased by PEGylation, the clearance through the kidneys would still be expected to be fairly rapid. PEG linkage is via thioester bond which can hydrolyze, although data show this to be very slow, and the data presented derives from the full PEG-CIPRO species detected by HPLC.

The CIPRO data, together with the free PEG and PEG-FITC data, support the claim that conjugation of PEG (≧1K) to a small molecule results in dominance of the physico-chemical characteristics by PEG such that the conjugate essentially behaves like PEG. Thus the pharmacokinetic behaviour of the small molecule can be (predictably) controlled by conjugation to PEG of sizes≧1K.

Example 21 Monodisperse PEG-Triamcinolone Molecules

In order to determine whether PEGylated compounds can cross a lipid bilayer and enter a cell, various monodisperse PEGS were conjugated to the corticosteroid tiamcinolone (TA). TA crosses the membrane rapidly so that any slowing effect of PEG conjugation could easily be measured.

To determine the location and movement of the PEGylated steroid, functional assays were planned which required the PEG moieties to be conjugated at sites in the steroid that would not inactivate the molecule. For corticosteroids, the 21′OH position is known to be essential for biological activity, rendering this site unsuitable for conjugation. However, modification of the hydroxyls at positions 16 and 17 allows retention, sometimes improvement, of biological activity, as demonstrated by the acetonide derivative of triamcinolone, formed by a reaction between acetone and a 1,2-diol. Based on this, it was reasoned that the steroid triamcinolone could be reacted with the mPEG aldehyde instead of a ketone to yield an acetal with the structure shown below. The conjugates containing the 3-mer PEG or 7-mer PEG were termed PEG-3-TA or PEG-7-TA respectively. The resulting molecular weights of PEG-3-TA and PEG-7-TA are 611 Da and 787 Da respectively. The structures of the parent triamcinolone and triamcinolone acetonide are depicted in below in C and D respectively.

A is the chemical structure of PEG-TA showing conjugation of PEG moiety at 16 and 17 positions of the steroid ring. B is the structure of the undesired “contaminant” showing attachment of the PEG at the 21′OH position, which will render this compound pharmacologically inactive. C is the structure of the parent steroid triamcinolone. D is the structure of triamcinolone acetonide, demonstrating modification at the 16 and 17 hydroxyls similar to that in PEG-TA.

For PEG-3-TA, the resulting reaction generated products in which the desired structure (A) constituted 95% of the final reaction product with the remaining 5% comprising the isomer depicted in B. For PEG-7-TA, the respective proportions were 90% and 10%. Since the isomeric contaminant contains a covalent linkage at the 21-OH position, this molecule will be inactive and thus not contribute to the overall biological activity. HPLC analysis demonstrated that there was no unreacted parent triamcinolone in the purified reaction product.

Example 22 Nuclear Translocation Assay of PEG-TA

To determine whether PEGylated steroids can enter cells, and to compare their potency with the parent steroid triamcinolone, cells expressing the human glucocorticoid receptor (hGR) were incubated with increasing concentrations of TA, PEG-3-TA and PEG-7-TA, and the subcellular location of the hGR determined using immunocytochemistry and fluorescence microscopy.

Chinese Hamster Ovary (CHO) cells were plated on glass coverslips in 24-well plates, and transfected with DNA encoding the hGR using 0.25 μg DNA per well. One day following transfection, cells were incubated with increasing concentrations, from 1 nM to 10 μM, of TA, PEG-3-TA or PEG-7-TA, overnight at 37° C.

Stock solutions of steroids were prepared at 2 mM (TA) or 1 mM (PEG-3-TA, PEG-7-TA) in ethanol. Each test compound was diluted to 10 μM into culture medium supplemented with dextran-coated charcoal treated serum (DCCS) (final 5% serum), and then further diluted to the final concentrations. This DCCS was employed to minimize background effects caused by steroids in the culture medium. Dilutions of steroid were calculated to ensure that cells were not exposed to ethanol concentrations greater than 1% (v/v). Previous studies using 1% ethanol confirmed that the solvent alone had no effect on the location of the hGR. Control, untreated cells were incubated with a 1:100 dilution of Ca²⁺/Mg²⁺-free PBS.

Following treatment with test compounds, cells were fixed in 3.7% (v/v) formaldehyde/PBS and permeabilized with 1% (v/v) Triton-X-100 in blocking buffer. Subcellular localization of the transfected hGR was determined by immunocytochemistry using an anti-GR antibody and a FITC-conjugated secondary antibody. Cells were counterstained with DAPI which binds the nuclei of all cells, transfected and untransfected. hGR localization was detected using fluorescence microscopy, and images taken with a CCD high resolution digital camera using multidimensional channel recording to allow acquisition of overlapping images from different fluorescence wavelengths.

FIG. 28 shows a dose-dependent nuclear translocation of the hGR induced by the three test compounds, TA, PEG-3-TA and PEG-7-TA, with a clear difference in potency between the compounds. The top panels show control samples containing untreated cells. The green stain spread across the whole cell is characteristic of cytosolic staining, with the nucleus somewhat discernible in the centre of the cell, and demonstrates that in the basal state, the hGR is almost entirely cytosolic.

In the presence of 1 nM TA, the hGR localizes predominantly in the nucleus, with a very low degree of staining remaining in the cytosol. Complete translocation to the nucleus occurs with TA concentrations at and above 10 nM. By contrast, the PEGylated forms of triamcinolone do not alter the hGR distribution at the lowest concentration. For PEG-3-TA, nuclear staining of the hGR begins to occur at 10 nM, and at 1 μM, all the transfected hGR detected is present in the nucleus. For PEG-7-TA, the translocating effect only begins to appear at 100 nM and is more clearly seen at 1 μM. At 10 μM, PEG-7-TA causes complete translocation of the transfected hGR from the cytosol to the nucleus.

The data show clearly that the PEGylated forms of triamcinolone are capable of inducing nuclear translocation of the hGR, albeit with much reduced efficiency compared with the parent steroid. This demonstrates that these compounds are capable of entering cells and of binding the receptor. Although this assay is not sufficiently quantitative to measure accurately the relative potency of the three compounds, it is clear that the order of potency is TA>PEG-3-TA>PEG-7-TA.

Example 23 Glucocorticoid Receptor Reporter Assay of PEG-TA

To quantitate the effect of PEGylation on the ability of the steroid to enter a cell and elicit a response, a glucocorticoid receptor reporter assay was employed. In this assay, treatment of cells with ligand leads to receptor-mediated expression of the firefly luciferase gene. The level of gene expression is quantitated by an enzymatic luciferase assay, and the amount of light produced is directly proportional to the expression level of the luciferase gene.

The results from the nuclear translocation assay demonstrate that conjugation of PEG at the 16- and 17-OH positions of the steroid ring impair, but do not prevent the drug from entering cells. Since TAA contains modifications at the 16 and 17 positions, it was chosen as the more appropriate comparator for PEG-TA in this quantitative assay.

Experimental Details

For these experiments, the Promega Dual Luciferase Reporter kit was employed. This involves a firefly luciferase reporter gene under the control of the Glucocorticoid Response Element (GRE-Luc), co-transfected into COS-7 cells with a gene encoding the hGR and a control vector containing a Renilla luciferase reporter gene under no promoter or enhancer elements (pRL-Null). The firefly luciferase gene is expressed only upon binding of a ligand-bound receptor to the GRE-Luc enhancer. The Renilla luciferase gene, by contrast, is constitutively expressed and can be assayed independently from the firefly luciferase, thus providing a control for variation in transfection efficiencies between samples. This is based on the commonly accepted observation that upon transfection with multiple plasmids, cells will generally either take up all the plasmids or no plasmid. Thus although transfection efficiency and cell numbers can vary between wells, cells transfected with the firefly luciferase gene will also contain the Renilla luciferase gene and the activity of the former can be normalized to that of the latter.

Cos-cells were plated in 96-well microplates and transfected with the relevant plasmids using standard procedures; all incubations and assays were performed in triplicate in these plates using medium containing DCCS, as described in section 8.3.1. Twenty-four hours post-transfection, cells were incubated with different concentrations of steroid for 20 hours. Cells were then lysed, and the expression levels of firefly and Renilla luciferase were determined by sequential enzymatic assays provided by the Promega Dual Luciferase Reporter Assay kit. The resulting luminescence was detected using the Tecan Genios Pro Microplate reader, and data were analyzed using GraphPad Prism 4.0 software.

Readings obtained in the absence of steroid represent “basal” background levels of luciferase expression. All readings in the presence of steroid are expressed as a ratio compared with this, and termed “fold increase over basal.” Values obtained for firefly luciferase were normalized to those obtained for Renilla luciferase in the corresponding well in order to compensate for differences in transfection efficiency or cell number.

In time-course experiments (e.g. FIG. 30), transfected cells were incubated with four different concentrations of steroid for increasing time periods. The time points at which test articles were applied to the cells were staggered to enable all reactions to terminate at the same time, thus allowing simultaneous assaying of all samples in a single microplate.

Results

Comparison of EC₅₀ values for TAA, PEG-3-TA, and PEG-7-TA revealed a marked difference in potency between the native and the PEGylated steroids. An EC₅₀ value represents the concentration of drug that produces a half-maximal response, and serves as the standard measure of comparing potencies of different compounds. Thus, a higher EC₅₀ value signifies a lower potency. FIG. 29 displays a representative experiment, showing a difference in EC₅₀ of 3 orders of magnitude between TAA and PEG-3-TA or PEG-7-TA. The figure demonstrates that the PEGylated steroids operate with a slightly lower efficacy (maximal response) but a dramatically reduced potency (EC₅₀) at these receptors. Comparative EC₅₀ values from a number of experiments are recorded in Table X, and reveal differences in potency from TAA of approximately 1300-fold and 5500-fold for PEG-3-TA and PEG-7-TA respectively.

To determine whether a similar difference is observed with the parent steroid, TA, which contains no modification at the 16- and 17-OH positions, the luciferase reporter assay was performed on TA.

TA displayed differences in EC₅₀ values of 150-fold and 630-fold respectively when compared with PEG-3-TA and PEG-7-TA, and was approximately 9-fold less potent than TAA. These data correlate well to the results obtained in the nuclear translocation assay, in which an approximate 2 orders of magnitude difference was noted between the PEGylated and parent forms of the steroid, and an additional difference found between the shorter (3-mer) and longer (7-mer) PEGylated derivatives.

To confirm that experiments were performed at equilibrium, time course experiments were performed. FIG. 30 demonstrates that by 20 h incubation, receptor activation values had reached their maximal levels and did not vary significantly for up to 48 h incubation, regardless of the concentration used. At early time-points, i.e. less than 6 h, submaximal activation was observed. This time course most likely reflects the lag time for the RNA transcription and protein synthesis that is required for the luciferase enzymes to be generated.

Verification that the PEG-TA derivatives were also assayed at equilibrium is found in the experiment (Luc-21) detailed in Appendix I, in which plates prepared in parallel were assayed after incubation with steroid for 24 or 48 h. The similar results produced in both plates confirm that reactions had reached equilibrium.

Example 24 Glucocorticoid Receptor Binding Assay of PEG-TA

To determine what component of the differences in receptor activation are due to the effect of PEG on receptor binding, a cell-free in vitro GR binding assay was employed. The assay is based on competitive inhibition binding of a fluorescently labeled high affinity ligand, and generates Ki values that can be compared between the test compounds. The Ki is a measure of the affinity of the binding interaction and derives from the concentration of competing ligand that produces 50% inhibition of maximal binding. Thus, as with EC₅₀ values, a higher Ki value reflects a weaker binding affinity.

A commercially available GR binding assay based on fluorescence polarization (FP) was utilized to compare the binding affinity of the PEGylated and native steroids (Pan Vera Glucocorticoid Receptor Competitor Assay). The assay is based on competitive inhibition and measures the ability of the test compound to compete with a fluorescently labeled ligand (Fluormone™ GS1) for binding to a purified preparation of the recombinant human glucocorticoid receptor.

In its free unbound form, the fluorescent ligand behaves as a small molecule with a high rotational velocity or “tumbling rate”, and correspondingly low ability to polarize fluorescence light (i.e. a low FP). When bound to receptor, the ligand essentially becomes part of a larger molecule, with a slower rotational velocity and a higher FP. Addition of competing ligand, the test compound, results in a dose-dependent decrease in FP as the fluorescent ligand is prevented from binding the receptor.

Standard competition assay procedures were employed. Briefly, serial dilutions of the test compound were incubated in triplicate wells of a black 96-well microplate together with the fluorescent ligand, and reactions were initiated by addition of purified recombinant receptor. Assays were incubated in the dark for 2-4 h, and then polarization values were read using the fluorescence polarization function of the Tecan Genios Pro microplate reader.

FIG. 31 shows results from a representative experiment and reveals a relatively modest difference in binding affinity between TA and the PEG-TA derivatives. In contrast to the large difference in potency observed in the luciferase assay, the cell-free binding assay produces differences in binding affinity of less than one order of magnitude. Average data from replicate experiments, detailed in Table X, produce a 4.2-fold and an 8.8-fold increase in Ki values for PEG-3-TA and PEG-7-TA respectively compared with TAA.

Interestingly, TA displayed an even lower binding affinity, with a Ki approximately 20-fold lower than that of TAA. The Ki value for dexamethasone was comparable to that of TAA, whereas that of a related corticosteroid, budesonide, appeared to be slightly lower.

TABLE X Assay REQUIRING entry to cell Luciferase assay TAA TA* PEG-3-TA PEG-7-TA EC₅₀ 0.062 ± 0.011 0.545 nM 81.7 ± 9.0 343.2 ± 54.2 nM nM nM Fold difference 1 8.8 1316 5533 vs TAA Assay INDEPENDENT of entry to cell Binding assay TAA TA** PEG-3-TA PEG-7-TA Ki 2.2 ± 0.5 nM 45.5 ± 21.1 nM 9.4 ± 2.4 nM 19.6 ± 5.1 nM Fold difference 1 20.3 4.2 8.8 vs TAA *(n = 1) **(n = 2) Data shown are EC₅₀ and Ki values ± standard error. TA values are based on 1 or 2 experiments as indicated. All other values are based on 5 or more experiments. Further details for these data can be found in Appendix I.

Except where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

The specification is most thoroughly understood in light of the teachings of the references cited within the specification, all of which are hereby incorporated by reference in their entirety. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan recognizes that many other embodiments are encompassed by the claimed invention and that it is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. 

1. A method of treating a patient in need of an opioid antagonist, comprising: administering to the patient an oral dosage form comprising a compound selected from the group consisting of 6-CH₃—(OCH₂CH₂)₅—O-naloxol; 6-CH₃—(OCH₂CH₂)₆—O-naloxol; 6-CH₃—(OCH₂CH₂)₇—O-naloxol; 6-CH₃—(OCH₂CH₂)₈—O-naloxol; and 6-CH₃—(OCH₂CH₂)₉—O-naloxol; or a pharmaceutically acceptable salt thereof, wherein the compound is an α-6 isomer, a β-6 isomer or a mixture of α-6 and β-6 isomers.
 2. The method of claim 1, wherein the compound comprises a mixture of α-6 and β-6 isomers.
 3. The method of claim 2, wherein the compound is 6-CH₃—(OCH₂CH₇)₇—O-naloxol or a pharmaceutically acceptable salt thereof.
 4. The method of claim 1, wherein the compound is selected from the group consisting of α-6-CH₃—(OCH₂CH₂)₅—O-naloxol; α-6-CH₃—(OCH₂CH₂)₆—O-naloxol; α-6-CH₃—(OCH₂CH₂)₇—O-naloxol; α-6-CH₃—(OCH₂CH₂)₈—O-naloxol; and α-6-CH₃—(OCH₂CH₂)₉—O-naloxol; or a pharmaceutically acceptable salt thereof.
 5. The method of claim 4, wherein the compound is α-6-CH₃—(OCH₂CH₂)₇—O-naloxol or a pharmaceutically acceptable salt thereof.
 6. The method of claim 1, wherein the compound is selected from the group consisting of β-6-CH₃—(OCH₂CH₂)₅—O-naloxol; β-6-CH₃—(OCH₂CH₂)₆—O-naloxol; β-6-CH₃—(OCH₂CH₂)₇—O-naloxol; β-6-CH₃—(OCH₂CH₂)₈—O-naloxol; and β-6-CH₃—(OCH₂CH₂)₉—O-naloxol; or a pharmaceutically acceptable salt thereof.
 7. The method of claim 1, wherein the compound is β-6-CH₃—(OCH₂CH₃)₇—O-naloxol or a pharmaceutically acceptable salt thereof. 